UNIVERSIDADE ESTADUAL DE SANTA CRUZ

PRÓ-REITORIA DE PESQUISA E PÓS-GRADUAÇÃO

PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E BIOLOGIA MOLECULAR

Estudos funcionais de genes da família de glutationa peroxidase e proteína de ligação ao

selênio de cacau envolvidos na interação cacau-Moniliophthora perniciosa

ÁKYLA MARIA MARTINS ALVES

ILHÉUS – BAHIA – BRASIL

Fevereiro de 2015

ÁKYLA MARIA MARTINS ALVES

Estudos funcionais de genes da família de glutationa peroxidase e selenium binding

protein de cacau envolvidos na interação cacau-Moniliophthora perniciosa

Dissertação apresentada ao Programa

de Genética e Biologia Molecular da

Universidade Estadual de Santa Cruz,

como parte das exigências para

obtenção do título de Mestre em

Genética e Biologia Molecular

ILHÉUS – BAHIA – BRASIL

Fevereiro de 2015

A474 Alves, Ákyla Maria Martins. Estudos funcionais de genes da família de glu- tationa peroxidase e proteína de ligação ao selê- nio de cacau envolvidos na interação cacau-Moni- liophthora perniciosa / Ákyla Maria Martins Alves. – Ilhéus, BA:UESC, 2015. vi, 69f. : Il. Orientadora: Fabienne Micheli. Dissertação (Mestrado) – Universidade Esta- dual de Santa Cruz. Programa de Pós-Graduação em Genética e Biologia Molecular. Inclui referências.

1. Cacaueiro – Doenças e pragas. 2. Vas- soura de bruxa (Fitopatologia). 3. Proteínas. 4. Selênio. I. Título.

CDD 633.74

ÁKYLA MARIA MARTINS ALVES

Estudos funcionais de genes da família de glutationa peroxidase e selenium binding

protein de cacau envolvidos na interação cacau-Moniliophthora perniciosa

Dissertação apresentada ao Programa

de Genética e Biologia Molecular da

Universidade Estadual de Santa Cruz,

como parte das exigências para

obtenção do título de Mestre em

Genética e Biologia Molecular.

APROVADA: 24 de fevereiro de 2015

Jane Lima

UESC

Fabienne Micheli

UESC/Cirad

Bruno Silva Andrade

UESB

Luciana Rodrigues Camillo

UESC

DEDICO

À comunidade científica, e em especial aos meus pais e todos os mestres que fizeram parte de minha

formação

“Penso noventa e nove vezes e nada descubro; deixo

de pensar, mergulho em profundo silêncio - e eis que

a verdade se me revela”.

Albert Einstein

AGRADECIMENTOS

A universidade Estadual de Santa Cruz - UESC.

A Fapesb pela concessão da bolsa.

A Dra. Fabienne Micheli pela orientação e apoio durante todas as etapas de realização do meu

mestrado. E também por ter se demonstrado sempre disponível a ajudar, pelo seu olhar crítico

e principalmente pelo seu profissionalismo ao me incentivar a fazer sempre o melhor e a

buscar novas perspectivas para realização dos trabalhos.

A todos os professores do Programa de Pós-Graduação em Genética e Biologia Molecular

(PPGGBM) da UESC em especial Professor Carlos Priminho pelo bom exemplo como

professor/pesquisador e aos Professores Leandro e Márcio pelo incentivo a leitura e escrita.

A professora Fátima Alvim pela orientação no estágio de docência.

A equipe que faz parte do colegiado do PPGGBM. Agradeço a Mara, Kátia e Fabrícia pela

disponibilidade em ajudar.

A toda equipe que fez parte da realização deste trabalho.

A toda equipe Biomol pelos momentos compartilhados: em especial a Sara pela

disponibilidade em ajudar, pela parceria e por sempre me aconselhar. A Laís pela ajuda

sempre que necessária durante a realização dos experimentos.

Ao Bruno Andrade e Gesivaldo por ter compartilhado tantos conhecimento comigo,

principalmente na área de modelagem molecular e pela boa recepção durante minhas visitas

ao laboratório de biologia computacional da UESB.

A todos os meus colegas de turma, pelos trabalhos realizados em conjunto, troca de

conhecimentos e pelo incentivo.

Agradeço a minha família por ter sempre incentivado e apoiado meus estudos e por ter

aceitado minha ausência em vários momentos durante meu mestrado.

Aos amigos conquistados aqui em Ilhéus: em especial a Carol por sua lealdade, pela

demonstração de carinho e cuidado, e pelo incentivo durante essa etapa, e a Maria por sua

generosidade.

A Alan pelo cuidado e incentivo durante a fase final desta etapa, principalmente durante a

redação do artigo. Pela demonstração de carinho, pela paciência e pelo companheirismo.

A todos que de alguma forma me ajudaram durante essa etapa e que torcem para que eu tenha

coragem de enfrentar novos desafios.

ÍNDICE

EXTRATO................................................................................................................... i

ABSTRACT................................................................................................................ iii

LISTA DE FIGURAS................................................................................................. v

LISTA DE TABELAS................................................................................................ vi

1. INTRODUCÃO....................................................................................................... 1

2. REVISÃO DE LITERATURA............................................................................... 4

2.1. A vassoura-de-bruxa do cacaueiro................................................................ 4

2.2. Morte celular programada em T. Cacao....................................................... 5

2.3. Importância do Selênio (Se) para os organismo........................................... 6

2.4. Modelagem de proteínas por homologia e docking molecular..................... 8

3. HIPÓTESE.............................................................................................................. 11

4. OBJETIVOS............................................................................................................ 11

4.1. Geral............................................................................................................. 11

4.2. Específicos................................................................................................... 11

5. CAPÍTULO I........................................................................................................... 12

5.1. Resultados................................................................................................... 12

5.2. Material e métodos...................................................................................... 13

5.2.1. Obtenção do gene TcSBP e elaboração da estratégia de clonagem... 13

5.2.2. Clonagem......................................................................................... 15

5.3. Conclusões................................................................................................... 15

5.4. Referencias.................................................................................................. 15

5.5. Anexos......................................................................................................... 19

6. CAPÍTULO II......................................................................................................... 22

7. CONCLUSÕES GERAIS....................................................................................... 69

i

EXTRATO

ALVES, Ákyla Maria Martins. Universidade Estadual de Santa Cruz, Ilhéus, Fevereiro de 2015.

Estudos funcionais de genes da família de glutationa peroxidase e selenium binding protein de

cacau envolvidos na interação cacau-Moniliophthora perniciosa. Orientadora: Dra. Fabienne

Micheli (UESC/Cirad). Co-orientadores: Márcio Gilberto Cardoso Costa (UESC) e Karina Peres

Gramacho (Ceplac).

O cacaueiro (Theobroma cacao L.) é uma planta endêmica das florestas tropicais da América do Sul e

é cultivada principalmente para a produção de licor de cacau, manteiga e pó para a indústria de

chocolate. No entanto, a produção de cacau tem sido severamente prejudicada por doenças causadas

por fungos e oomicetos. No Brasil, a doença vassoura-de-bruxa, causada pelo basidiomiceto

Moniliophthora perniciosa provocou drásticas mudanças econômicas e sociais nas áreas afetadas.

Devido à importância econômica do cacau, estratégias que visem à elucidação dos mecanismos da

interação entre o T. cacao e M. perniciosa são indispensáveis, como por exemplo, compreender a

relação entre genes diferencialmente expressos durante essa interação através de estudos funcionais in

silico e in vitro. Aqui foi estudada a expressão gênica e realizado estudo computacional de três

proteínas de cacau denominadas, proteína de ligação ao selênio (selenium-binding protein, SBP),

glutationa peroxidase de hidroperóxidos fosfolipídicos (PHGPx) e glutationa peroxidase 2 (GPx2).

Estas proteínas já foram caracterizadas em diferentes organismos e estão relacionadas com a função

imunológica desses, devido suas relações com compostos de selênio. Dessa forma, objetivamos

caracterizar in silico e modelar por homologia a SBP, PHGPx e GPX2 de T. cacao, buscar um ligante

para SBP e realizar ensaios de interação proteína-proteína entre SBP-PHGPx/GPX2. Além disso,

analisamos a expressão desses genes durante a interação cacau-M. perniciosa. Através de buscas no

banco de dados do genoma do cacau (CocoaGenDB) foram encontradas as sequências dos genes SBP,

PHGPx e GPX2 que codificam para proteínas com 476, 239 e 262 aminoácidos, respectivamente. As

sequências foram comparadas com banco de dados públicos através da ferramenta BLASTP e foram

reveladas identidades proteicas em diferentes espécies. Foram então realizados alinhamentos múltiplos

por meio do ClustalW para identificar regiões conservadas entre a sequências, e em seguida foram

construídas árvores filogenéticas da TcSBP e de toda família de GPXs de cacau usando o software

Mega6. Foi demonstrado que a TcSBP e TcGPXs de cacau apresenta homologia com essa classe de

proteínas em mamíferos e plantas. A modelagem das proteínas alvos foi realizada através do programa

Swiss Model online. Foram utilizadas como molde a estrutura da SBP de Sulfolobus tokodaii a qual

apresentou 38% de identidade com a TcSBP, e estrutura da GPX5 de Populus trichocarpa que

apresentou 63,92% de identidade com TcPHGPx e 67,72% de identidade com TcGPx2. A qualidade

estereoquímica dos modelos foi avaliada através dos programas Procheck e Anolea. A estrutura

química de compostos de Se foram obtidas do banco de dados PubChem para posteriormente realizar

o docking com a TcSBP através das ferramentas do Autodock Vina. O docking entre o TcSBP e o

ii

selenito ocorreu no motivo conservado CSSC com uma energia de -1.9 kcal/mol. O docking entre

TcSBP-TcPHGPX e TcSBP-TcGPx2 foram realizados através da ferramenta Rosetta (rosettadock). A

visualização da modelagem e do docking foi possível usando o PyMol e o Discovery Studio 4.0. A

análise da expressão de TcSBP, TcPHGPx e TcGPx2 em meristemas de plântulas de cacau variedades

Catongo (suscetível) e TSH1188 (resistentes a M. perniciosa), inoculadas ou não com M. perniciosa,

foi obtida por RT-qPCR. A expressão relativa foi analisada com o método de Ct comparativo (2-ΔΔCt)

usando como referência os genes endógenos actina e malato desidrogenase, e como calibrador as

plantas não-inoculadas (controle). Os dados obtidos foram submetidos à análise de variância

(ANOVA), seguido pelo teste de separação de médias Scott-Knott p(p≤0.01). As análise de RT-qPCR

em plantas susceptíveis de cacau infectados por M. perniciosa mostrou que, no período de 30 a 90

dias, durante a fase de estabelecimento da doença vassoura-de-bruxa o gene TcSBP foi

significativamente mais expressa do que TcPHGPx e TcGPx2. Enquanto o gene TcGPx2 na variedade

resistente é superexpesso nas fase inicial e final da doença. Para realização da clonagem do gene

TcSBP foram desenhados primers específicos com os quais a sequência foi amplificada. Em seguida, o

produto de amplificação foi purificado, digerido com as enzimas NdeI e HindIII, e ligado no vetor

pET-28a. A reação de ligação foi utilizada para transformação de Escherichia coli BL21 (DE3);

colônias transformadas foram identificadas por PCR. Pretende-se futuramente realizar ensaios de

indução da proteína SBP para posteriores testes in vitro. Todos os passos deste trabalho serão

importantes para a compreensão da relação dos genes SBP, PHGPX e GPx2 com a resistência do T.

cacao contra M. perniciosa, além disso, este estudo trará uma nova perspectiva que vise compreender

a interação funcional e física das proteínas codificadas por esses genes em uma espécie vegetal.

Palavras-chave: Theobroma cacao; vassoura-de-bruxa; selênio; proteína de ligação ao selênio;

glutationa peroxidase.

iii

ABSTRACT

ALVES, Ákyla Maria Martins. Universidade Estadual de Santa Cruz, Ilhéus, February of

2015. Expression and functional studies of the glutathione peroxidase family and

selenium binding-protein from cacao involved in the cacao-Moniliophthora perniciosa

interaction. Advisor: Dra. Fabienne Micheli (UESC/Cirad). Co-advisores: Márcio Gilberto

Cardoso Costa (UESC) and Karina Peres Gramacho (Ceplac).

The cacao tree (Theobroma cacao L.) is a plant endemic from the tropical forests of South

America and is mainly cultivated for the production of cacao liquor, butter and powder for the

chocolate industry. However, the production of cacao was highly prejudiced by diseases

caused by fungus and oomycetes. In Brazil, the witches’ broom disease, caused by the

basidiomycete Moniliophthora perniciosa triggered drastic economic and social changes in

the affected areas. Due to the economic importance of the cacao, strategies focusing the

elucidation of the interaction mechanisms between T. cacao and M. perniciosa appeared as

necessary, as, for example, the understanding of the relation between differentially expressed

genes during this interaction or through in silico and in vitro functional studies. Here, we

developed expression analysis and computational studies of three cacao proteins named

selenium binding protein (SBP), phospholipid hydroperoxide glutathione peroxidase

(PHGPx) and glutathione peroxidase 2 (GPx2). These proteins already have been

characterized in different organisms and are related with their immunological functions, due

to their relations with selenium compounds. Thus, the aim of this study was the in silico

characterization and the homology modeling of SBP, PHGPx and GPX2 from T. cacao, the

search of SBP ligand, and the realization of in silico docking assays SBP-PHGPx/GPX2.

Moreover, we analyzed the expression of these genes during the cacao-M. perniciosa

interaction. Through data mining from cacao genome database (CocoaGenDB), sequences of

SBP, PHGPX and GPX2 genes that encode for proteins of 476, 239 and 262 amino acids,

respectively, were found. The sequences were compared with public databanks using the

BLASTP tool and protein identity was observed in different species. Then, we realized

multiple alignment using the ClustalW program to identify conserved regions between

sequences, and, then, phylogenetic trees were built for TcSBP and for the all GPX family

from cacao using the Mega6 software. We showed that the TcSBP and TcGPXs from cacao

presented high homology with these class of proteins in plants and mammalians. The

modeling of the target proteins was realized using the Swiss Model program. The structure of

the SBP from Sulfolobus tokodaii, which presented 38% of identity with TcSBP, and the structure of

iv

GPX5 from Populus trichocarpa, which presented 63.92% of identity with TcPHGPx and 67.72%

with TcGPx2 were used as molds. The stereochemical quality of the molds was evaluated through the

Procheck and Anolea programs. The chemical structure of the Se compound was obtained from the

PubChem database for subsequent realization of the docking of SBP using the Autodock Vina tool.

The docking between TcSBP and the selenite occurred at the conserved CSSC motif with a -1.9

kcal/mol configuration. The docking between TcSBP-TsPHGOX and TcSBP-TcGPx2 were realized

using the Rosetta server (rosettadock). The visualization of the modeling and docking was made using

the PyMol and Discovery Studio 4.0. The analysis of the expression of TcSBP, TcPHGPX and

TcGPx2 in plantlet meristems of susceptible (Catongo) and resistant (TSH1188) cacao varieties

inoculated or not with M. perniciosa, was obtained by RT-qPCR. The relative expression was

analyzed with the comparative Ct method (2-ΔΔCt) using actin and malate dehydrogenase as reference

endogenous genes, and non inoculated plants (control) as calibrator. The data were submitted to

vairance analysis (ANOVA), followed by the Scott-Knott test p(p≤0.01). The analyse of RT-qPCR in

susceptible cacao infected by M. perniciosa showed that, from 30 to 90 days, during the beginning of

the witches’ broom disease, the TcSBP gene was significantly more expressed than TcPHGPX and

TcGPx2. While the TcPGx2 gene was overexpresses in the resistant variety in the initial and final

disease phases. For the cloning of the TcSBP gene, specific primers were designed and used to amplify

the sequence. Then the amplification product was purified, digested with NdeI and HindIII enzymes,

and cloned into the pET-28a vector. The ligation reaction was used for Escherichia coli BL21

(DE3) transformation; transformed colonies were identified by PCR. As perspectives, it is

planned to realize the SBP protein induction assay for posterior in vitro tests. All the steps of

this work are important to understand the relation of the SBP, PHGPX and GPx2 with the

resistance of T. cacao against M. perniciosa, and this study will lead to new perspectives

aiming the understanding of the functional an physical interaction of the proteins encoded

these genes in a plant species.

Keywords: Theobroma cacao; witches’ broom disease, selenium, selenium-binding protein;

glutathione peroxidase.

v

LISTA DE FIGURAS

Figura 1. Os sintomas da doença vassoura-de-bruxa.................................................................. 6

Figura 2. A rotação é permitida em torno das ligações N–Cα e Cα–C............................ 11

CAPITULO 1.

Figura 1. Vetor pET-28a (+)....................................................................................................... 62

Figura 2. Amplificação do gene SBP a partir do cDNA de T. cacao......................................... 63

Figura 3. PCR de colônia para confirmação da transformação de E. coli com o vetor pET-

28a (+) contendo o inserto de interesse (gene TcSBP)................................................

64

CAPITULO 2.

Figura 1. Structural features of SBP gene and glutathione peroxidase gene family of

T.

Xcacao............................................................................................................

42

Figura 2. Phylogenetic analysis, using amino acid data, of TcSBP and SBP from other

species………………………………………………………………………...

43

Figura 3. Phylogenetic relationship between the amino acid sequences of TcGPXs and others

plants and mammalian GPXs………………………………………………………

44

Figura 4. Tridimensional structure of TcSBP, TcPHGPx and TcGPx2 obtained by homology

modeling……………………………………………………………………………..

45

Figura 5. The conserved motifs of TcSBP and TcGPx2………………………………………. 46

Figura 6. Docking between TcSBP and selenite, TcSBP and TcGPx2, and TcSBP and

TcPHGPX…………………………………………………………………….

47

Figura 7. Relative expression of TcSBP, TcPHGPx and TcGPx2 in cacao meristems

inoculated or not (control) with M. perniciosa……………………………….

48

vi

LISTA DE TABELAS

Tabela 1. Gene and protein characteristics of SBP and GPX family from T. cacao 38

1

1. INTRODUÇÃO

O cacaueiro (Theobroma cacao L.) é uma planta diploide (2n = 20) (FIGUEIRA et

al., 1992) endêmica das florestas tropicais da América do Sul que foi domesticada há

aproximadamente 3.000 anos atrás, na América Central (WOOD GAR, 1985). Esta espécie é

de grande importância econômica, pois o seu cultivo está relacionado principalmente ao

aproveitamento de suas sementes para produção de licor, manteiga e pó de cacau, matérias

primas fundamentais para a indústria de chocolates. Contudo, derivados e subprodutos do

cacau podem também serem transformados em cosméticos, bebidas finas, geleias, sorvetes e

sucos, dentre outros produtos (GESTEIRA et al., 2007; LORENZI; MATOS, 2002).

No mundo são produzidas cerca de 4 milhões de toneladas de cacau por ano,

distribuídos entre os continentes africano, americano e asiático (INTERNATIONAL COCOA

ORGANIZATION, 2014). No entanto, doenças causadas por fungos, oomicetos e vírus, bem

como as pragas ocasionadas por insetos, são responsáveis por cerca de 40% das perdas de

colheita de cacau na América do Sul e nas ilhas do Caribe (INTERNATIONAL COCOA

ORGANIZATION, 2014; AIME; PHILLIPS-MORA, 2005). No Brasil, por exemplo, o

surgimento da doença vassoura-de-bruxa, causada pelo fungo hemibiotrófico Moniliophthora

perniciosa ocasionou uma drástica queda na produção anual de cacau (AIME; PHILLIPS-

MORA, 2005).

Antes da ocorrência da vassoura-de-bruxa o Brasil era o segundo maior produtor de

cacau do mundo, com cerca de 350 mil toneladas/ano (SEBRAE, 2014). Após a entrada do

fungo M. perniciosa, ocorreu uma redução na produtividade nacional seguida de grandes

prejuízos econômicos nas regiões produtoras, o que fez nosso país despencar no ranking da

produção mundial, passando de exportador para importador de amêndoas (MEINHARDT et

al., 2008).

Na Bahia, onde a primeira incidência da doença foi observado em 1989 (GARCIA et

al, 2007; GRIFFITH et al, 2003), a devastação da vassoura-de-bruxa tem sido considerada a

mais drástica do que em qualquer uma das outras regiões infectadas, em parte devido à alta

densidade de fazendas de cacau na região da floresta de Mata Atlântica, mas também por ser

uma região que há ausência de uma estação seca distinta, o que favorece a dispersão dos

esporos durante todo o ano. Estima-se que 200 000 pessoas foram afetadas diretamente e mais

de dois milhões de pessoas até hoje estão sendo afetados indiretamente, devido aos impactos

2

sociais, econômicos e ambientais gerados (GRIFFITH et al, 2003; INTERNATIONAL

COCOA ORGANIZATION, 2013).

Dessa forma, vários esforços têm sido deflagrados no sentido de recuperar a

cacauicultura, por meio de métodos de controle que incluem o manejo e poda fitossanitária

(remoção das partes infectadas) e lançamento de novas variedades (PURDY; SCHIMDT,

1996). As ferramentas de genômicas e proteômicas, em conjunto com a biologia molecular e

ferramentas computacionais, vêm se tornando assim, cada vez mais indispensáveis para

compreensão de mecanismos envolvidos na interação cacau-M. perniciosa, na perspectiva de

obtenção de plantas resistentes.

Um importante trabalho identificou genes diferencialmente expressos envolvidos em

eventos biológicos relacionados à defesa de plantas de cacau contra o M. perniciosa por meio

de análise do perfil de expressão de ESTs (Expressed Sequence Tags) (GESTEIRA et al.,

2007; DA HORA JÚNIOR et al., 2012). Foram identificados, por exemplo, que os genes que

codificam para selenium-binding protein (TcSBP) e glutationa peroxidases (TcGPX) são

diferencialmente expressos durante interação cacau-M. perniciosa (GESTEIRA et al., 2007).

Estes genes são conhecidos pela alta homologia entre as espécies e estão envolvidos com os

mecanismos de morte celular programada (PCD) (FANG et al., 2010; ZHANG et al., 2013).

Esse mecanismo segundo Ceita et al. (2007) ocorre de forma gradativa durante a infecção do

cacau por M. perniciosa beneficiando a mudança de fase biotrófica para necrotrófica do

fungo.

Nossa investigação foi então realizada utilizando duas diferentes abordagens, a

primeira foi fundamentada em estudos in silico buscando a caracterização da TcSBP e das

proteínas da família TcGPX. Em mamíferos essas proteínas são conhecidas pela capacidade

de induzir apoptose (SBP) (FANG et al., 2010; ZHANG et al., 2013) e inibir apoptose (GPX)

(FRANCO; CIDLOWSKI, 2009) e apresentam alta homologia com espécies de plantas (WU

et al., 2010; RAMOS et al., 2009). As análises in silico partiram assim, da busca por

homologia entre sequências de aminoácidos para implicação estrutural e funcional. Na

segunda abordagem realizamos análise de expressão gênica de TcSBP e dois genes da família

TcGPx, a que codificam para glutationa peroxidase de hidroperóxidos fosfolipídicos

(TcPHGPx) e para glutationa peroxidase 2 (TcGPx2).

Este trabalho foi divido em duas partes: na primeira, mostramos os resultados da

clonagem e expressão do gene TcSBP em sistema heterólogo para posteriormente realizar

indução desta proteína e assim testá-la in vitro em estudos futuros. A segunda parte foi

3

desenvolvida na forma de artigo científico cuja estrutura foi organizada visando uma futura

submissão na revista BioMed Central journals (BMC). Nele apresentamos os dados de análise

de expressão e caracterização in silico genes e proteínas glutationa peroxidases e proteína de

ligação ao selênio envolvidos na interação cacau-M. perniciosa. Nossa perspectiva é em

realizar investigações mais aprofundadas sobre a importância dessas proteínas durante a

indução da morte celular programada (PCD) de T. cacao durante a patogênese causada pelo

M. perniciosa. Almejamos assim encontrar alvos moleculares anti-apoptóticos que

possibilitem o manejo da doença vassoura-de-bruxa através da diminuição dos efeitos

causados pela infecção de M. perniciosa em cacau, permitindo uma menor susceptibilidade.

4

2. REVISÃO DE LITERATURA

2.1. A vassoura-de-bruxa do cacaueiro

A doença vassoura-de-bruxa do cacau é resultante da interação entre T. cacao e o

fungo hemibiotrófico M. perniciosa. Essa doença ocorre em vários países como, por exemplo,

Bolívia, Brasil, Peru, Venezuela, Suriname, Guiana, Colômbia e Equador (PURDY;

SCHIMDT, 1996). Na Bahia, onde a primeira incidência da doença foi observada em 1989 a

ocorrência da vassoura-de-bruxa foi devastadora, devido à região oferecer as condições ideais

de desenvolvimento para o fungo (PURDY; SCHIMDT, 1996; ROCHA; WHEELER, 1985).

A dispersão do fungo causador da vassoura-de-bruxa se dá principalmente pelo vento

e pela chuva, e a altura em que é produzido é importante para o progresso da doença e a

umidade relativa do ar próxima à saturação e temperaturas entre 20 e 30 ºC são as condições

ideais (ROCHA; WHEELER, 1985). A vassoura-de-bruxa se desenvolve em duas fases

distintas, uma biotrófica e outra necrotrófica. Na fase biotrófica, o M. perniciosa cresce de

forma intercelular e utiliza os nutrientes derivados do apoplasto da planta durante um período

prolongado (30 a 60 dias) (FRIAS et al., 1991; TEIXEIRA et al., 2014).

Durante fase biotrófica o M. perniciosa tem uma grande capacidade de degradar

pectina na planta facilitando assim a sua infecção e colonização. O subproduto dessa

degradação é o metanol que pode ser fonte de carbono para o crescimento do M. perniciosa

durante a fase de vassoura verde (Fig.1a) (OLIVEIRA et al. 2012). Estudos têm demostrado

que durante a fase biotrófica o M. perniciosa não suprime totalmente as defesas da planta

(TEIXEIRA eta al., 2014) como é observado por exemplo, para o agente patogénico

biotrófico Oidium neolycopersici (WASPI et al, 2001). O Colletotrichum graminicola fungo

causador da antracnose de cereais é um análogo de M. perniciosa que também não suprimi

totalmente as defesas da planta e usa essa estratégia para causar avirulência durante a infecção

(VARGAS et al, 2012). Essa estratégia contribui para o desenvolvimento da fase necrotrófica

e morte dos tecidos vegetais (TEIXEIRA et al., 2014).

A segunda fase, saprotrófica é caracterizada pela mudança do fungo para fase

saprofítica, com a difusão intracelular do micélio dicariótico, o qual causa necrose e morte do

tecido distal do ponto original de infecção, formando a vassoura seca (Fig.2b) favorecendo

assim o fungo M. perniciosa (LEAL et al., 2010).

5

Figura 1. Os sintomas da doença vassoura-de-bruxa. (a) vassoura verde; (b) vassoura seca; (c)

almofada floral infectada; (d) fruto infectado. Fonte: Griffith 2004.

Essa mudança de fase ocorre devido ao colapso de tecidos infectados, e um processo

de senescência. Após os primeiros sinais de senescência de cacau, o fungo

mata ativamente os tecidos de cacau remanescentes. Como consequência, os nutrientes

solúveis derivados de células hospedeiras mortas se tornam disponíveis a partir das hifas

necrotróficas, que mais tarde produzem basidiomas, completando assim o ciclo da doença

(TEIXEIRA et al., 2014).

Dessa forma, o ciclo de vida do M. perniciosa induz sintomas que dependem do

estágio da doença. O crescimento hipertrófico e hiperplasia dos tecidos, a perda de

dominância apical, e proliferação de rebentos axilares, resulta na formação de hastes anormais

(vassouras verdes) (Fig. 1a). A infecção de almofadas florais (Fig.2c) leva ao surgimento de

lançamentos vegetativos, a produção de flores anormais e frutos partenocárpicos (Fig.2d). O

ciclo de vida do M. perniciosa se completa com a formação de vassouras secas,

consequentemente os frutos também apresentam aspectos seco e os basidiósporos são

dispersos na atmosfera (PURDY; SMITH, 1996).

2.2. Morte celular programada em T. Cacao

A morte celular programada (PCD) é um processo ativo, controlado geneticamente no

qual as células são eliminadas seletivamente em um ambiente altamente coordenado por

6

proteases e nucleases específicas. Durante esse processo apenas as células que estão

destinadas a morrer são destruídas e nenhuma outra célula vizinha é atingida (PETROV et al.,

2015). Esse mecanismo é similar entre os diferentes organismos (ALBERTS, 2010) e ocorre

pelos processos de quebra do citoplasma, condensação nuclear, aumento da permeabilidade de

membrana, influxo de cálcio, liberação do citocromo C, ativação de proteases específicas e

fragmentação do DNA (ALBERTS, 2010; GRIFFITHS, et al., 2001).

Em plantas normais a PCD é vital para o seu crescimento e desenvolvimento. Por

outro lado, PCD pode ser uma consequência de estresses bióticos e abióticos graves. Dessa

forma, PCD induzida por estresse afeta significativamente a produtividade de plantas de

fundamental importância para a agricultura (MITTLER; BLUMWALD, 2010).

Durante a interação planta-patógeno a morte celular é ativada através do processo de

Resposta de Hipersensibilidade (HR) da planta que se caracteriza por uma resposta rápida

ocorrida no sítio de infecção levando a formação de uma lesão localizada, delimitada por

tecido saudável, visando inibir a dispersão do agente invasor (CORDEIRO; SÁ, 1999). Em T.

cacao foi demonstrado que durante a infecção pelo M. perniciosa são desencadeados eventos

de PCD importantes, os quais levam ao desenvolvimento da doença nos tecidos da plantas.

Também há relatos da formação de cristais de oxalato de cálcio em células do tecido de

cacaueiro infectado com M. perniciosa ao longo do desenvolvimento da doença,

principalmente nas fases mais tardias (cerca de 90 dias após a infecção (CEITA et al., 2007).

Durante a mudança de fase do M. perniciosa de biotrófica para necrotróficas

aminoácidos são convertidos em amidas no tecido da planta do cacau (SCARPARI et al.,

2005). Além disso, o crescimento intracelular do fungo também esta associado ao acúmulo de

peróxido de hidrogênio (DIAS et al., 2011). Dessa forma, estes processos seriam um sinal

fisiológico para a indução de PCD o que poderia ser interpretado como um esforço fisiológico

da planta em mobilizar nutrientes dos tecidos degenerados.

2.3. Importância do Selênio (Se) para os animais e vegetais

O selênio (Se) foi descoberto em 1817 por Jons Jacob Berzelius. Este elemento traço

está localizado no grupo 16 da tabela periódica e compartilha propriedades físico-químicas

com o enxofre (S). Esta similaridade permite a substituição do Se por S em diversas reações

químicas que ocorrem nos sistemas biológicos (MCKENZIE et al., 1998). As fontes ricas de

Se incluem castanha-do-pará, grãos, frutos do mar, fígado e outras carnes. No entanto, os

níveis de Se no solo determinam o conteúdo de Se nas formas inorgânicas, selenito e selenato,

7

e orgânicas, como o aminoácido selenometionina (SeMet) e selenocisteína (SeCis) nos

vegetais. A SeMet é um aminoácido que contém Se integrado no lugar do átomo S na

molécula de metionina, enquanto, a SeCis contém um átomo de Se no lugar do S da cisteína.

Estes aminoácidos são incorporados nas proteínas dos organismos formando as

selenoproteínas com importantes funções biológicas (VIARO; VIARO, 2001; AGALOU et

al., 2005). As SeCis são codificadas de uma maneira especial por um códon UGA, que

normalmente é um códon de parada, enquanto as SeMets são codificadas a partir do mesmo

códon da metionina, AUG. Assim as SeMet não são reconhecidas como uma espécie de Se

durante sua incorporação em proteínas (SUZUKI, 2005). As principais selenoproteínas são as

glutationa peroxidases, iodotironina deiodinases e as tiorredoxina redutases (MCKENZIE al.,

1998). Estas selenoproteínas estão presentes nas células, e as alterações na sua expressão

explicam muitas das manifestações clínicas e bioquímicas de deficiência de Se. Em 1957,

com a descoberta da glutationa peroxidase foi comprovado que o Se está envolvido na função

imunológica (MCKENZIE al., 1998; VIARO; VIARO, 2001).

Em humanos e animais há várias deficiências imunitárias e doenças que resultam de

uma inadequada ingestão dietética de Se (MCKENZIE al., 1998). Foi relatado que em

determinadas regiões da China, por exemplo, onde os solos são pobres neste mineral, a

ingestão reduzida de Se está relacionada com o desenvolvimento de doenças como cardiopatia

juvenil (Keshan) e degeneração das articulações (Kashin-Beck). Na Finlândia, a elevada

prevalência da miopatia do músculo esquelético em animais também foi associada aos baixos

níveis de Se no solo. Estudos sobre a incidência de câncer e AIDS indicam que áreas onde o

solo é muito rico em Se as taxas destas doenças são reduzidas (MCKENZIE al., 1998). No

entanto, é importante considerar que níveis de Se acima do esperado podem causar efeitos

tóxicos nas células (MCKENZIE al., 1998; AGALOU et al., 2005). O Se também é conhecido

pelo seu efeito antitumoral e esse efeito está associado a uma proteína denomina proteína de

ligação ao selênio que tem a capacidade de se ligar ao selênio e desencadear o mecanismo de

apoptose em células cancerígenas. Estudos recentes evidenciaram que há uma correlação

entre os níveis de expressão de SBP1 em células de câncer e a sobrevida de pacientes, o que

demonstra o papel funcional desta proteína na proliferação de linhagens destas células e do

efeito anti-tumoral de Se (AGALOU et al., 2005; ZHANG et al., 2013).

Existem poucos relatos sobre a função das SBPs em plantas, mas resultados obtidos

apontam que os níveis de expressão de SBPs estão envolvidos em processos de

desintoxicação, e na resistência a estresse biótico (AGALOU et al., 2005; SAWADA et al.,

8

2004). Um estudo com Arabidopsis thaliana demonstrou que há uma correlação entre os

níveis de expressão de AtSBP1 e a tolerância a toxicidade causada pelo selenito, o que indica

uma função deste gene para desintoxicação celular nesta espécie. Esta função é importante,

pois algumas plantas são sensíveis a altos níveis de Se levando a inibição do crescimento,

clorose, secagem das folhas, e consequentemente morte da planta (AGALOU et al., 2005).

Também em Arabidopsis ficou evidenciado que a expressão de AtSBP1 está relacionado com

condições de estresse, e que esse gene pode estar envolvido no processo de desintoxicação

causada pelo cádmio (DUTILLEUL et al., 2008). Devido as SBPs serem proteínas altamente

conservadas e encontradas em muitos organismos é sugerido um compartilhamento do papel

biológico destas proteínas entre as diferentes espécies (FLEMETAKIS et al., 2002). No

entanto, poucos estudos foram realizados em espécies animais e vegetais.

2.4. Modelagem de proteínas por homologia e docking molecular

Com o desenvolvimento de técnicas moleculares e das ômicas em paralelo com o

avanço dos estudos na área da bioinformática esforços têm sido feitos em todo o mundo, por

instituições governamentais e privadas, no sentido de elucidar o maior número possível de

estruturas tridimensionais de proteínas. Dentre as técnicas de bioinformática, a modelagem

molecular vem sendo usada para a construção de moléculas e executar um variedade de

cálculos a fim de prever suas características químicas. Dessa forma, a modelagem molecular

engloba todos os métodos teóricos e técnicas computacionais usados para modelar ou imitar o

comportamento de moléculas (NORWELL; MACHALEK, 2000; SANTOS FILHO et al.,

2002).

Para cada uma das etapas no processo de modelagem por homologia existe um grande

número de métodos, programas e servidores. O GenBank por exemplo é mais conhecido

banco de sequências primárias (National Center for Biotechnology Information – NCBI). O

banco de dados de estruturas proteicas (Protein Data Bank – PDB) é essencial para a

modelagem, pois é o repositório para estruturas de cristais de biologia macromolecular

(WESTBROOK et al., 2000).

Com a disponibilidade destas estruturas no PDB, a modelagem por homologia,

também conhecida como modelagem comparativa se tornado cada vez mais utilizada. Esta

abordagem baseia-se em alguns padrões gerais que têm sido observados, em nível molecular,

no processo de evolução biológica: (a) homologia entre sequências de aminoácidos implica

9

em semelhança estrutural e funcional; (b) proteínas homólogas apresentam regiões internas

conservadas; (c) as principais diferenças estruturais entre proteínas homólogas ocorrem nas

regiões externas, constituídas principalmente por alças (“loops”), que ligam os elementos de

estruturas secundárias. Outro fato importante é que as proteínas agrupam-se em um número

limitado de famílias tridimensionais. Estima-se que existam cerca de 5.000 famílias proteicas.

Consequentemente, quando se conhece a estrutura de pelo menos um representante de uma

família, é geralmente possível modelar, por homologia, os demais membros da família

(BRANDEL; TOOZE, 1991; SANTOS FILHO et al., 2002).

Os métodos correntes de modelagem de proteínas por homologia implicam

basicamente em quatro passos sucessivos: (a) identificação e seleção de proteínas-molde; (b)

alinhamento das sequências de resíduos; (c) construção das coordenadas do modelo; (d)

validação da estrutura alvo modelada. Se o grau de identidade entre as estruturas primárias

das proteínas-molde e da proteína-alvo for igual ou superior a 25%, quando o número de

resíduos é superior a 80, existe grande probabilidade de que estas proteínas tenham estruturas

tridimensionais semelhantes e pode-se construir um modelo (SANDER, 1991).

A qualidade estereoquímica do modelo é de importância fundamental. O programa

mais utilizado na avaliação dos parâmetros estereoquímicos, o PROCHECK, avalia os

comprimentos de ligação, os ângulos planos, a planaridade dos anéis de cadeias laterais, a

quiralidade, as conformações das cadeias laterais, a planaridade das ligações peptídicas, os

ângulos torcionais da cadeia principal e das cadeias laterais, o impedimento estérico entre

pares de átomos não ligados e a qualidade do gráfico de Ramachandran. O gráfico de

Ramachandran é particularmente útil porque ele define os resíduos que se encontram nas

regiões energicamente mais favoráveis e desfavoráveis e orienta a avaliação da qualidade de

modelos teóricos ou experimentais de proteínas (RAMACHANDRAN; SASISEKHARAN,

1968).

Figura 2. A rotação é permitida em torno das ligações N –Cα e Cα –C. Fonte: Lehninger et

al. (2002).

10

Quando se tem uma proteína alvo modelada por homologia, uma nova abordagem

pode ser usada para avaliar a afinidade dessa molécula com outras, sejam elas orgânicas ou

inorgânicas através de metodologias computacionais de docking receptor-ligante. Dessa

forma Docking pode ser definido como um problema “chave-fechadura”, que possui a

finalidade de prever os modos de interação entre duas moléculas conhecendo apenas suas

estruturas tridimensionais isoladas. Assim, um método docking deve ser capaz de prever

corretamente a conformação do ligante, e as interações físico-químicas associadas (GUEDES

et al, 2014).

11

3. HIPÓTESE

As proteínas TcSBP e TcGPXs são importantes para a regulação do ciclo celular em

Theobroma cacao. A TcSBP induz apoptose e participa do mecanismo de morte celular

programada (PCD) e TcGPx inibe apoptose dificultando assim o desenvolvimento da doença

vassoura-de-bruxa.

4. OBJETIVOS

4.1 Geral

Analisar a expressão e caracterizar in silico genes e proteínas glutationa peroxidases (GPXs) e

proteína de ligação ao selênio (SBP) envolvidos na interação cacau-M. perniciosa.

4.2. Específicos

1. Caracterizar in silico o gene TcSBP e os genes da família TcGPX;

2. Modelar por homologia a SBP, PHGPx e GPX2 de T. cacao;

3. Buscar compostos inorgânicos de selênio que interagem com TcSBP;

4. Realizar docking molecular entre proteínas;

5. Analisar a expressão dos genes TcPHGPx, TcGPx2 e SBP entre plantas resistentes e

susceptíveis inoculadas versus não inoculadas com M. perniciosa;

6. Realizar clonagem do gene TcSBP e expressá-lo em bactéria para posteriores estudos

funcionais.

12

5. CAPÍTULO I

5.1. Resultados

O teste de amplificação do gene TcSBP com o primers específicos foi realizado

em cDNA sintetizado a partir do produto de RNA extraído de meristemas de T. cacao

da variedade Catongo inoculado com M. perniciosa. Foram utilizadas temperaturas de

anelamento entre 52°C e 60°C, sendo que a 58°C, foi obtida uma melhor amplificação

do gene. O padrão de banda do produto da reação de PCR foi analisado em gel de

agarose e foi verificado que a banda amplificada possui o tamanho esperado para o gene

TcSBP (1500 pb; Fig. 2). Apenas uma banda foi visualizada no gel o que possibilitou a

realização de uma purificação direta do produto de PCR (sem cortar previamente a

banda do gel). A eletroforese em gel de agarose permitiu a separação, identificação e

purificação do cDNA de TcSBP.

Figura 2. Amplificação do gene SBP a partir do cDNA de T. cacao. (M) Marcador 1 kb

(Invitrogen). (1) controle negativo. (2) e (3) Produto de amplificação (aproximadamente 1500

pb).

Após purificação, o cDNA de TcSBP foi clonado no vetor pET-28a (+) para

transformação de E. coli estirpe BL21A reação de PCR de colônias foi feita e o produto

da reação analisado em eletroforese em gel de agarose, permitindo a identificação de

vários clones positivos para o gene TcSBP (Fig.3).

13

Figura 3. PCR de colônia para confirmação da transformação de E. coli com o vetor pET-28a

(+) contendo o inserto de interesse (gene TcSBP). (M) Marcador 1kb (Invitrogen) (1 a 3).

Produtos de amplificação obtidos a partir das colônias bacterianas.

5.2. Material e métodos

Os experimentos foram realizados no Laboratório de Biologia Molecular

localizado no Centro de Biotecnologia e Genética (CBG).

5.2.1. Obtenção do gene TcSBP e elaboração da estratégia de clonagem

Meristemas de plântulas de T. cacao variedade Catongo foram inoculados com o

fungo M. perniciosa, e coletados entre 24 horas e 90 dias congeladas no nitrogênio

líquido e conservadas no freezer -80°C. O material vegetal foi macerado no nitrogênio

líquido e utilizado para extração de RNA. O RNA total foi extraído utilizado o

RNAqueous Kit/Plant RNA Isolation Aid de acordo com as recomendações do

fornecedor (Ambion). A qualidade e integridade do RNA foram avaliadas por análise

em gel de agarose 1,5%. O cDNA dupla fita foi sintetizado utilizando o Super Script

Double Stranded cDNA Synthesis kit seguindo as recomendações do fabricante

(Invitrogen®). Em seguida o cDNA foi submetido a reação de amplificação utilizando

primers específicos do gene TcSBP (número de acesso no GenBank: XP 007034202.1).

Os primers foram desenhados em função da sequencia TcSBP e da estratégia de

clonagem para expressão em sistema heterólogo. Para isso, foram utilizados os

programas Custom Primers-OligoPerfect™ Designer (Invitrogen), OligoAnalyser 3.1

14

(IDT SciTools/Integrated DNA Tecnologies, Inc. – Califórnia, EUA), e Webcutter

(http://rna.lundberg.gu.se/cutter2/) para obtenção de uma mapa de restrição (Anexo I) e

desenho dos primers que permitissem uma clonagem direta no vetor pET-28a (+) (Fig.

1) sem a necessidade de realização de uma subclonagem.

Os primers desenhados foram: F 5’-AAGACAGGGCATATGGCCGGTAACG -

3’ e R 5’- CGGTAAAGAGTGAAGCTTCACAGCAAG -3’ com sítio de restrição para

Nde I e Hind III respectivamente. A reação de amplificação foi realizada em volume

total de 25 μl contendo 1 μl da amostra de cDNA, 2,5 μl de tampão da PCR 10X, 0,75

µl de cloreto de magnésio (1,5mM), 1 μM de cada oligonucleotídeo (0,2 mol/ μl), 1U de

Taq DNA Polimerase Recombinante (Invitrogen) e água (qsp 25 µl). A reação foi

conduzida em termociclador e consistiu nas seguintes etapas: 4 minutos a 94ºC

(desnaturação inicial); 30 segundos a 94ºC (desnaturação); 1 minuto a 58ºC (anelamento

dos primers); 4 minutos a 72ºC (extensão); 7 minutos a 72ºC (extensão final); espera

final de 15ºC. O produto de PCR foi analisado um gel de agarose a 1% corado com 1 µl

de GelRED. A purificação do produto de PCR foi realizada utilizando o kit de

purificação de DNA (Qiagen – QIAquick Gel Extraction Kit Protocol) de acordo com

recomendações do fabricante.

Figura 1. Vetor pET-28a (+). Fonte: pET System Manual (Novagen).

15

5.2.2. Clonagem

O produto de PCR purificados do gel foi submetido à digestão em reação de

volume final de 50 µl contendo com 8 μl de DNA purificado, 10 μl do tampão (Tango),

1 μl da enzima NdeI, 1 μl da enzima Hind III e 30 μl de água. A reação foi incubada a

37°C por 4h. O vetor pET-28a (+) também foi digerido com 3 μl da enzima NdeI e 3 μl

da enzima Hind III. A reação foi incubada a 37°C por 2h em banho-maria. Em seguida,

foram adicionados 2 µl de enzima CIAP à reação, e qual foi incubada por 30 min a

37°C. Após a enzima CIAP foi inativada a 85 ºC por 15 min foi realizada a purificação

com o kit (Qiagen). A reação de ligação foi realizada com 9 μl de inserto, 1 μl do

plasmídeo, 1 μl da enzima T4-DNA ligase e 1,5 µl de tampão 10X. O produto de

ligação foi utilizado para transformação por choque térmico de Escherichia coli BL21.

Após a transformação de E. coli e crescimento de colônias nas placas com meio seletivo

contendo canamicina a 50 µg/ml e cloranfenicol a 25 µg/ml, foi realizada uma reação

de PCR de colônias utilizando o protocolo descrito no §4.1. Sendo o controle negativo o

produto da reação sem amostra de DNA. As reações de PCR de colônia foram

analisadas em gel de eletroforese de agarose 1% corado com 1 µl Gel Red.

5.3. Conclusões

O gene TcSBP foi amplificado a 58ºC;

E. coli foi transformado com o vetor pET-28(a) contendo o gene TcSBP.

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5.5. Anexos

Anexo I - Mapa de restrição do gene TcSBP Enzyme No. Positions Recognition

name cuts of sites sequence

AatI 1 460 agg/cct

Acc65I 1 637 g/gtacc

AccB1I 1 637 g/gyrcc

AccB7I 1 932 ccannnn/ntgg

AccIII 1 1800 t/ccgga

AcsI 3 573 1729 1858 r/aatty

AflIII 1 1586 a/crygt

Alw21I 1 683 gwgcw/c

AocI 1 436 cc/tnagg

ApoI 3 573 1729 1858 r/aatty

Asp700I 1 702 gaann/nnttc

Asp718I 1 637 g/gtacc

AspHI 1 683 gwgcw/c

BalI 1 370 tgg/cca

BanI 1 637 g/gyrcc

BanII 1 683 grgcy/c

BbsI 1 457 gaagac

Bbv12I 1 683 gwgcw/c

Bbv16II 1 457 gaagac

BclI 1 661 t/gatca

20

BpiI 1 457 gaagac

BpuAI 1 457 gaagac

BsaAI 1 380 yac/gtr

BsaBI 1 211 gatnn/nnatc

BsaI 1 1376 ggtctc

BsaWI 1 1800 w/ccggw

Bse118I 1 60 r/ccggy

Bse21I 1 436 cc/tnagg

Bse8I 1 211 gatnn/nnatc

BseAI 1 1800 t/ccgga

BsgI 1 1468 gtgcag

Bsh1365I 1 211 gatnn/nnatc

BshNI 1 637 g/gyrcc

BsiHKAI 1 683 gwgcw/c

BsiI 2 1442 1873 ctcgtg

BsiMI 1 1800 t/ccgga

Bsp13I 1 1800 t/ccgga

Bsp1407I 1 754 t/gtaca

Bsp19I 1 314 c/catgg

BspEI 1 1800 t/ccgga

BspLU11I 1 1586 a/catgt

BsrBRI 1 211 gatnn/nnatc

BsrDI 2 420 1186 gcaatg

BsrFI 1 60 r/ccggy

BsrGI 1 754 t/gtaca

BssAI 1 60 r/ccggy

BssSI 2 1442 1873 ctcgtg

BssT1I 3 314 541 817 c/cwwgg

BstDSI 1 314 c/crygg

BstSFI 5 199 685 1147 1341 1568 c/tryag

BstX2I 4 212 320 791 1198 r/gatcy

BstYI 4 212 320 791 1198 r/gatcy

Bsu36I 1 436 cc/tnagg

Cfr10I 1 60 r/ccggy

CfrI 2 58 368 y/ggccr

CvnI 1 436 cc/tnagg

DraII 2 1082 1224 rg/gnccy

DsaI 1 314 c/crygg

EaeI 2 58 368 y/ggccr

Eam1104I 2 40 180 ctcttc

EarI 2 40 180 ctcttc

Ecl136II 1 681 gag/ctc

Eco130I 3 314 541 817 c/cwwgg

Eco147I 1 460 agg/cct

Eco24I 1 683 grgcy/c

Eco31I 1 1376 ggtctc

Eco32I 1 1449 gat/atc

Eco57I 3 330 716 959 ctgaag

Eco64I 1 637 g/gyrcc

Eco81I 1 436 cc/tnagg

EcoICRI 1 681 gag/ctc

EcoO109I 2 1082 1224 rg/gnccy

EcoRI 1 573 g/aattc

EcoRV 1 1449 gat/atc

EcoT14I 3 314 541 817 c/cwwgg

EcoT22I 1 1617 atgca/t

ErhI 3 314 541 817 c/cwwgg

Esp1396I 1 932 ccannnn/ntgg

FbaI 1 661 t/gatca

FriOI 1 683 grgcy/c

HincII 2 1355 1400 gty/rac

HindII 2 1355 1400 gty/rac

Kpn2I 1 1800 t/ccgga

KpnI 1 641 ggtac/c

Ksp22I 1 661 t/gatca

Ksp632I 2 40 180 ctcttc

MamI 1 211 gatnn/nnatc

MflI 4 212 320 791 1198 r/gatcy

MluNI 1 370 tgg/cca

Mph1103I 1 1617 atgca/t

MroI 1 1800 t/ccgga

MscI 1 370 tgg/cca

MslI 6 748 1057 1645 1712 1723 1816 caynn/nnrtg

MspA1I 1 760 cmg/ckg

NcoI 1 314 c/catgg

NsiI 1 1617 atgca/t

NspBII 1 760 cmg/ckg

21

NspI 4 720 1340 1590 1619 rcatg/y

PflMI 1 932 ccannnn/ntgg

Pme55I 1 460 agg/cct

Ppu10I 1 1613 a/tgcat

PpuMI 2 1082 1224 rg/gwccy

Psp124BI 1 683 gagct/c

Psp5II 2 1082 1224 rg/gwccy

PstI 1 689 ctgca/g

PvuII 1 760 cag/ctg

SacI 1 683 gagct/c

SfcI 5 199 685 1147 1341 1568 c/tryag

SseBI 1 460 agg/cct

SspBI 1 754 t/gtaca

SspI 1 1076 aat/att

SstI 1 683 gagct/c

StuI 1 460 agg/cct

StyI 3 314 541 817 c/cwwgg

Van91I 1 932 ccannnn/ntgg

XhoII 4 212 320 791 1198 r/gatcy

XmnI 1 702 gaann/nnttc

Zsp2I 1 1617 atgca/t

The following endonucleases were selected but don't cut this sequence:

AatII, Acc113I, Acc16I, AccBSI, AccI, AclNI, AcyI, AfeI, AflII, AgeI, AhdI,

Alw44I, AlwNI, Ama87I, Aor51HI, ApaI, ApaLI, AscI, AseI, AsnI, AspEI, AspI,

AtsI, AvaI, AviII, AvrII, BamHI, BanIII, BbeI, BbiII, BbrPI, BbuI, BcgI,

BcoI, BfrI, BglI, BglII, BlnI, BlpI, BpmI, Bpu1102I, Bpu14I, Bsa29I, BsaHI,

BsaMI, BsaOI, BscI, BseCI, BsePI, BseRI, Bsh1285I, BsiEI, BsiWI, BsmBI,

BsmI, BsoBI, Bsp106I, Bsp119I, Bsp120I, Bsp143II, Bsp1720I, Bsp68I, BspCI,

BspDI, BspHI, BspMI, BspTI, BspXI, BsrBI, BssHII, Bst1107I, Bst98I, BstBI,

BstD102I, BstEII, BstH2I, BstI, BstMCI, BstPI, BstSNI, BstXI, BstZI, Bsu15I,

CciNI, CelII, Cfr42I, Cfr9I, ClaI, CpoI, Csp45I, CspI, DraI, DraIII, DrdI,

EagI, Eam1105I, EclHKI, EclXI, Eco105I, Eco255I, Eco47III, Eco52I, Eco72I,

Eco88I, Eco91I, EcoNI, EcoO65I, EheI, Esp3I, FauNDI, FseI, FspI, GsuI,

HaeII, Hin1I, HindIII, HpaI, Hsp92I, KasI, KspI, LspI, MfeI, MluI, MroNI,

Msp17I, MspCI, MunI, Mva1269I, NaeI, NarI, NdeI, NgoAIV, NgoMI, NheI, NotI,

NruI, NspV, PacI, PaeI, PaeR7I, Pfl23II, PinAI, Ple19I, PmaCI, PmeI, PmlI,

PshAI, PshBI, Psp1406I, PspAI, PspALI, PspEI, PspLI, PspOMI, PstNHI, PvuI,

RcaI, RsrII, SacII, SalI, SapI, SbfI, ScaI, SexAI, SfiI, Sfr274I, Sfr303I,

SfuI, SgfI, SgrAI, SmaI, SmiI, SnaBI, SpeI, SphI, SplI, SrfI, Sse8387I,

SstII, SunI, SwaI, Tth111I, Vha464I, VneI, VspI, XbaI, XcmI, XhoI, XmaI,

XmaIII

22

6. CAPÍTULO II

The selenium-binding protein and glutathione peroxidase family of Theobroma

cacao: In silico analysis, homology modeling and gene expression analysis under

biotic stress conditions

Ákyla Maria Martins Alves1, Sara Pereira Menezes1, Eline Matos Lima1, Karina Peres

Gramacho2, Bruno Silva Andrade3, Fabienne Micheli1,4,*

1Universidade Estadual de Santa Cruz (UESC), Departamento de Ciências Biológicas

(DCB), Centro de Biotecnologia e Genética (CBG), Rodovia Ilhéus-Itabuna, km 16,

45662-900 Ilhéus-BA, Brazil.

2Cocoa Research Center, CEPLAC/CEPEC, 45600-970 Itabuna-BA, Brasil.

3Universidade Estadual do Sudoeste da Bahia (UESB), Av. José Moreira Sobrinho,

Jequié, Bahia, 45206-190, Brazil

4CIRAD, UMR AGAP, F-34398 Montpellier, France

*Corresponding author: Dr Fabienne Micheli, UESC, DCB, Rodovia Ilhéus-Itabuna

km16, 45662-900, Ilhéus-BA, Brazil. Phone: +55 73 3680 5196. Fax: +55 73 3680

5226. E-mail: fabienne.micheli@cirad.fr

23

Abstract

Background. The selenium-containing proteins are important for immune function of

various organisms and are highly conserved. The selenium-binding protein (SBP) has

selenium affinity. The selenoproteins are known to have selenium in the form of

selenocysteine (SeCys) in polypeptide chain eg. glutathione peroxidase. The glutathione

peroxidase family are most selenoproteins with the exception of phospholipid

hydroperoxide glutathione peroxidase (PHGPx) of plants which has a cysteine in place

of the active site instead of a SeSys. The relationship of these genes with the response to

biotic or abiotic stresses in plants has been studied and it is known which are crucial to

the defense mechanisms mainly due to antioxidant function. Important information is

that is known that in human, SBP induces apoptosis while GPx inhibits apoptosis. This

was the first study in silico and genic expression that analyzed the relationship of these

genes together during biotic stress in plants. The TcSBP and TcGPXs were identified

from a cacao-Moniliophthora perniciosa interaction cDNA library. Results. In the

cocoa genome database (DB CocoaGen) was found only a sequence encoding for

TcSBP and five TcGPXs. Multiples phosphorylation sites were found during the

prediction of post-translational events. Multiple alignments revealed conserved domains

in plants and human. TcSBP presents a CSSC, DELHH and HGD domains. The

TcGPXs have FPCNQF domain which has a conserved cysteine in place of the active

site. Homology modeling of three proteins of cacao was performed and used as template

the structure the SBP of Sulfolobus tokodaii which showed 38% identity and structure

the GPX5 of Populus trichocarpa which showed 63.92% identity with TcPHGPx and

67.72% identity with TcGPX2. After prediction of conserved domains and homology

modeling was performed docking analysis. The TcSBP has affinity with selenite and

TcGPx2 in active site CSSC and other site with TcPHGPx. RT-qPCR analysis in

24

susceptible plants of cocoa infected by M. perniciosa showed that in the period from 30

to 90 days when there is the establishment phase of the witches' broom disease TcSBP

gene (inducer of apoptosis) was significantly more expressed than TcPHGPx and

TcGPx2 genes. While in the resistant variety TcGPx2 gene (inhibitor of apoptosis) is

overexpressed during the asymptomatic phase and significantly more expressed than

TcSBP and TcPHGPx genes during the establishment of the dry broom.

Conclusion. To our knowledge this is the first report of physical interaction of SBP and

GPX in plant species. The knowledge of 3-D structure of the TcSBP, TcPHGPx and

TcGPx2 was important for identifying the possible biochemical function. The data

obtained in the in silico analysis and by RT-qPCR indicate a possible functional

interaction between TcSBP and TcGPXs. Furthermore, such genes are differentially

expressed during cacao-M. perniciosa interaction. But the TcSBP gene it is not

indicated for the induction of resistance by overexpression. This because programmed

cell death is a mechanism by which M. perniciosa changes to dikaryotic phase.

However use of genes that inhibit apoptosis, e.g. TcGPXs may be a good candidate for

obtaining cacao plants resistant to M. perniciosa.

Keywords: witches’ broom disease, selenium, molecular docking, gene expression

25

Introduction

Selenium (Se) is a trace element essential to many organisms including some archaea,

bacteria, protozoan, green algae, and nearly all animals, but it is non-essential in land

plants [1-3]. The richest sources of Se include Brazil nut, grains, seafood, liver and

other meats [4]. The daily Se requirement in human adult is 60 to 70 mg, and the plants

are considered as the main Se source of the human dietary [5]. However, if the intake of

Se in human exceed 400 mg, this compound becomes toxic [6, 7]. In plants, this

compound is present under both inorganic (selenite) and organic forms

(selenomethionine [SeMet] and selenocysteine [SeCys]) [3, 8]. The Se shared physico-

chemical properties with the sulfur (S) and the similarity of these properties allows the

replacement of S by Se in various chemical reactions that occur in biological systems

[1, 9]. For example, amino acids containing Se integrated in place of the S atom in

methionine molecule (SeMet), or containing Se in place of S in cysteine (Secys), may

be incorporated in proteins, forming selenoproteins [8-10]. The SeCys is encoded in a

special way by a UGA codon – which is a stop codon – while the SeMet is encoded by

the methionine codon, AUG [8, 9].

Selenoproteins have an important immune function in several species [11] and

one of the major classes of selenoproteins is the glutathione peroxidases (GPXs; EC

1.11.1.9) family that corresponds to antioxidant enzymes [12, 13]. The GPXs reduce

H2O2 and organic hydroperoxides to water and to the corresponding alcohols, using

reduced glutathione (GSH). GPXs inhibit the ROS-induced damage of membrane and

protein, and play a crucial role by protecting cells from oxidative damage [13, 14]. The

phospholipid hydroperoxide glutathione peroxidase (PHGPX; EC 1.11.1.12) is a

member of the GPX family, and is responsible for the direct reduction of phospholipid

hydroperoxide and lipid hydroperoxide. Even if PHGPXs are selenoproteins, they differ

from the other GPX by the presence of a cysteine as catalytic residue in their active site,

26

instead of selenocysteine [15]. Several studies have been conducted on GPX genes in

different plant species such as Nicotiana sylvestris [16], Citrus sinensis [17],

Lycopersicon esculentum [18], Arabidopsis thaliana [3, 15, 19], Lotus japonicas [20],

Oryza sativa [14] and Panax ginseng [21]. However, even if some plant GPX have been

isolated and characterized, only few genome-wide GPX family identification and

characterization studies have been reported [13].

Another category of selenium containing proteins are the selenium-binding

proteins (SBPs) which have the ability to bind covalently Se, but did not have any

seleno-amino acid incorporated into their polypeptide chain [22]. There were some

reports on the role of SBPs in plants, showing that SBP expression was related to

resistance to abiotic and biotic stresses [23, 24]. In transgenic rice plants (Oryza sativa),

the overexpression of OsSBP increased the plant resistance to the rice blast disease

caused by the fungus Pyricularia grisea, and to the bacterial blight due to Xanthomonas

oryzae pv. oryzae [23]. In Arabidopsis thaliana, increased expression of AtSBP1

confers tolerance to toxicity caused by selenite, suggesting that this gene could play a

role of detoxification [24].

The SBP and GPXs genes have been extensively investigated in animals and

human, generally in related studies [9, 25]. In human, it has been reported that the

expression of the SBP gene was reduced in tumor tissues when compared to healthy

ones, and for this reason, this gene has been indicated as a good predictor of clinical

outcome of cancer [10, 26]. Moreover, the level of SBP gene from human (HsSBP1)

was antagonist to the expression level of the human GPx1 (HsGPx1), i.e. the HsSBP1

gene expression decreased when the expression of HsGPx1 increased. The HsGPx1

gene was associated to several diseases, including cancer due to disease risk-associated

alleles [9, 25, 27]. Several studies have also reported that HsSPB and HsGPx1 proteins

27

were involved in cell cycle regulation, inhibiting (in the case of GPX) or inducing (in

the case of SBP) apoptosis, and interacting functionally and physically together [9, 25,

27, 28]. In Theobroma cacao L., SBP and GPX genes were identified from a cacao-

Moniliophthora perniciosa interaction cDNA library and/or from the cacao genome [29,

30]. Cacao is an endemic plant of tropical forests in South America [31] and is grown

mainly for the production of cocoa liquor, butter and powder for the chocolate industry

[32]. However, cocoa production has been severely damaged by diseases caused by

fungi and oomycetes [33]. In Brazil, the witches' broom disease caused by the

basidiomycete M. perniciosa caused drastic economic and social changes in the affected

areas [34]. Moniliophthora perniciosa has two distinct phases of development: a

biotrophic phase characterized by a monokaryotic and intercellular mycelium, and a

necrotrophic phase characterized by a dikaryotic and intracellular mycelium [35, 36].

The transition from biotrophic to necrotrophic phase is characterized by significant

accumulation of H2O2 and programmed cell death (PCD). The production of calcium

oxalate causes the production of reactive oxygen species (ROS) and therefore the PCD.

In cacao plants susceptible to M. perniciosa, the PCD occurs initially in the plant as a

defense mechanism and then is deflected by the fungus for its own profit, allowing

sporulation and further propagation [35]. Thus, the presence of compounds, genes and

proteins that induce apoptosis, facilitates the transition from biotrophic to necrotrophic

phase [35-37]. Thus the study of genes and proteins that may be involved in this

mechanism are important for better understand the cacao-M. perniciosa pathosystem

and so develop strategies for witches' broom disease control.

Here, we identified one SBP and five GPX sequences from T. cacao and we

analyzed in silico and in vitro three of them (TcSBP, TcPHGPX and TcGPx2).

Modeling and docking analyses revealed that TcSBP interacts with Se and, individually,

28

with TcPHGPX and TcGPx2. The putative role of TcSBP and TcGPX as inducer and

repressor of apoptose, respectively, was shown during the cacao-M. perniciosa

interaction by the RT-qPCR. In susceptible cacao plants infected by M. perniciosa,

TcSBP was significantly expressed during the establishment disease. While in the

resistant cacao variety infected by M. perniciosa, TcGPx2 is highly expressed at the

beginning of the asymptomatic phase and at the final times of the infection. To our

knowledge this is the first in silico study and expression analysis of both SBP and GPX

genes during biotic stress in plants and the first analysis of these genes in the T. cacao-

M. perniciosa interaction. This study shows that TcSBP and TcGPXs are important

targets to understand both M. perniciosa infection mechanism, and cacao defense

against this fungus. The use of genes that inhibit apoptosis, e.g. TcGPXs, may be a good

strategy to obtain cacao plants resistant to M. perniciosa.

Results

In silico analysis of SBP and GPX family of T. cacao

In silico analysis on CocoaGenDB revealed the presence of a unique sequence encoding

TcSBP and five sequences encoding TcGPXs (Fig.1). TcSBP was located on the

chromosome 4, while the TcGPX genes were located on four different chromosomes

(chromosome 1/TcGPX4, chromosome 3/TcGPX6 and TcGPX8, chromosome

5/TcPHGPX and chromosome 9/TcGPX2; Fig.1). The TcSBP gene is 4774 bp in length,

contains 7 exons and 6 introns (Fig.1) and has an ORF of 1431 bp (Table 1). TcSBP

encodes a protein of 476 amino acids, with a MW of 52732.7 Da and a putative pI of

5.74 (Table 1). Twenty one putative phosphorylation and no glycosylation sites were

observed in the TcSBP protein sequence (Table 1).

29

TcSBP does not contain any signal peptide, and its subcellular location is unknown

(Table 1). The alignment of TcSBP with SBPs from other organisms revealed high

identity with plant species and human and allowed the identification of the CSSC,

DELHH and HGD conserved region (Supplementary material 1).

The data mining of the CocoaGenDB allowed the identification of one

TcPHGPx and four TcGPXs (TcGPx2, TcGPx4, TcGPx6 and TcGPx8) (Fig. 1). The

TcPHGPx gene was 2581 bp in length, contained 6 exons and 5 introns (Fig. 1) and had

an ORF of 720 bp (Table 1). TcPHGPx encoded a protein of 239 amino acids, with a

molecular weight of 26477.2 Da and a putative pI of 9.08. Fourteen putative

phosphorylation and one glycosylation sites were found (Table 1). The TcPHGPx was

predicted to be located in the chloroplast (Table 1). The TcGPx2 gene was 2320 bp in

length, contained 6 exons and 5 introns (Fig. 1) and had an ORF of 789 bp (Table 1).

TcGPx2 encoded a protein of 262 amino acids, with a molecular weight of 29708.3 Da

and a putative pI of 9.11. Twelve phosphorylation and two glycosylation sites were

predicted (Table 1). The TcGPx2 was predicted as secreted (Table 1). The TcGPx4 gene

was 4805 bp in length, contained 6 exons and 5 introns (Fig. 1), and had an ORF of 171

bp (Table 1). TcGPx4 encoded a protein of 171 amino acids, with a molecular weight of

19175.9 Da and a putative pI of 9.14 (Table 1). Seven phosphorylation and three

glycosylation sites were predicted (Table 1). The subcellular location of TcGPx2 was

unknown (Table 1). The TcGPx6 gene was 2222 bp in length, contained 6 exons and 5

introns (Fig. 1), and had an ORF of 780 bp (Table 1). TcGPx6 encoded a protein of 235

amino acids, with a molecular weight of 25973.7 Da and a putative pI of 8.93 (Table 1).

Twenty-two phosphorylation and 3 glycosylation sites were predicted (Table 1).

TcGPx6 was predicted to be located in the chloroplast (Table 1). The TcGPx8 gene was

3189 bp in length, contained 6 exons and 5 introns (Fig. 1), and had an ORF of 573 bp

30

(Table 1). TcGGPx8 encoded a protein of 190 amino acids, with a molecular weight of

21600.4 Da and a putative pI of 5.27 (Table 1). Thirteen phosphorylation and 1

glycosylation sites were predicted (Table 1). The subcellular location of TcGPx8 was

unknown (Table 1). The alignment of TcGPXs sequence using the BLASTP tool

revealed high identity with GPXs from other organisms (plant and human), and allowed

the identification of the FPCNQF conserved region containing a conserved cysteine

residue (Supplementary material 2). The full-length amino acid sequences of plants and

mammalian SBPs and GPXs available in the databases were used to build an unrooted

phylogenetic tree (Fig. 2 and 3; Supplementary material 3 and 4). Plant and mammalian

SBPs were separated and formed two distinct branches in the tree (Fig. 2). Plant and

mammalian GPXs were separated and also formed two distinct branches in the tree

(Fig. 3). The five TcGPxs have closest phylogenetic relationship with members of the

same classification in other plant species (Fig.3).

Homology modeling of TcSBP, TcPHGPx and TcGPx2

The modeling of the TcSBP was based in the X-ray structure of the SBP from

Sulfolobus tokodaii (StSBP). The three-dimensional (3D) structure of the TcPHGPx and

TcGPx2 was based in the X-ray structure of the reduced form of the poplar glutathione

peroxidase (PtGPX5). The alignment of the amino acid sequence of TcSBP with the

StSBP (Fig. 4a) presented 38% of identity. The alignment of the amino acid sequence of

TcPHGPx and TcGPx2 with the PtGPx5 (Fig. 4b) presented 63.92% and 67.72% of

identity, respectively. The structural modeling of TcSBP revealed the presence of 7 α-

helices and numerous β-strands and P-loops (Fig. 4c). The structural modeling of

TcPHGPx and TcGPx2 were highly similar and presented, respectively, 5 and 7 α-

helices, 5 and 6 β-strands and several loops (Fig. 4d and e). The Ramachandran plots

31

showed that 99.9% of the amino acids of the TcSBP model were located in the most

favored regions and that 100% of the amino acids of the TcPHGPx and TcGPx2 models

were located in the most favored regions (Supplementary material 5). The conserved

motifs DELHH and HGD of TcSBP were located at the surface of the protein in two

distinct locations (Fig. 5a and b), while the CSSC motif that correspond to the putative

catalytic site of the protein was located inside the protein (Fig. 6a and b). The conserved

domain FPCNQF of TcGPx2, containing the conserved cysteine residue and

corresponding to the catalytic site, formed a cavity in which the cysteine residue was

found (Fig. 5c and d).

Molecular docking of TcSBP with selenite, TcGPx2 and TcPHGPX

The interaction between the conserved motif CSSC of TcSBP (Fig.6a and b) and the

selenite (Fig.6c) occurred with a setting of -1.9 kCal/mol. No interaction between

TcSBP and selenate was observed (data not shown). The results obtained by molecular

docking between proteins showed that TcSBP could interact both with TcGPx2 (Fig.6d)

and TcPHGPx (Fig.6e). The interaction between TcSBP and TcGPx2 occurred near to

the CSSC motif (Fig.6d) while the interaction between TcSBP and TcPHGPX occurred

in a region distant from the CSSC motif (Fig.6e).

Differential expression of TcSBP, TcGPx2 and TcPHGPX in resistant and

susceptible cacao genotypes infected by M. perniciosa

The expression of the TcSBP, TcPHGPx and TcGPx2 genes was analyzed together for

each cacao genotype, i.e. TSH1188 (resistant to witches’ broom disease) and Catongo

(susceptible), infected or not (control) with M. perniciosa (Fig. 7). For both genotypes

32

and for all the harvesting point, the PCR amplification occurred at the same and unique

melting temperature for each gene showing that only the corresponding gene was

amplified (Supplementary material 6). The RT-qPCR analysis in Catongo variety

showed that in the period from 24 hai (asymptomatic phase) TcSBP gene expression

was significantly higher than the one of TcPHGPx and TcGPx2, which were less

expressed than the control (Fig. 7a). At 48 hai, the expression of TcSBP did not differ

significantly from the one of TcPHGPx and control, but was significantly higher than

the expression of TcGPx2 (Fig. 7a). At 72 hai, these genes were significantly less

expressed than the control, and their expression differed between them (Fig. 7a). At 8

dai, the expression of TcGPx2 was higher than the TcSBP one. At 15 dai, TcPHGPx

was more expressed than TcSBP and TcGPx2. Through the period from 30 to 90 dai,

when occurred the establishment phase of the witches' broom disease, TcSBP was more

expressed than TcPHGPx and TcGPx2. Moreover, in the same period, the TcPHGPx

was highly repressed. Generally, the tendency was that when TcSBP was more

expressed, TcGPx2 is less expressed or repressed. The same trend occurs between

TcSBP and TcPHGPx (Fig.7a). In TSH1188, the same tendency was also observed (Fig.

7b). When TcGPx2 was more expressed, TcSBP is less expressed (Fig.7b). The TcGPx2

gene was overexpressed during the asymptomatic phase, and significantly more

expressed than TcSBP (24 and 48 hai). At 72 hai (swelling phase), TcGPx2 was also

significantly more expressed than TcSBP and TcPHGx. At 8 dai, TcSBP was more

expressed than TcPHGPx and TcGPx2. At 15 dai (green broom phase), TcGPx2

returned to be significantly more expressed than TcSBP. At 45 and 90 dai, TcGPx2

maintaining a higher expression than TcSBP (Fig.7b).

33

Discussion

In this article, we characterized by in silico analysis a SBP and five GPX (TcPHGPx,

TcGPx2, TcGPx4, TcGPx8 and TcGPx8) genes from T. cacao (Fig. 1). All the

corresponding proteins contained multiple predicted phosphorylation sites (Table 1).

Protein phosphorylation is a ubiquitous mechanism for the temporal and spatial

regulation of proteins involved in almost every cellular process [38]. TcSBP contained

three domains, DELHH, HGD and CSSC, also highly conserved among the SBPs from

other organisms (Fig.5a,b and 6a,b). It has been suggested that the characteristic CSSC

motifs have a strong affinity for selenium and heavy metals [22, 39-41]. In T. cacao, we

observed that the conserved domain CSSC was able to bind, in silico, the selenite (Fig.

6c) and potentially may be a binding site for other metals as observed in other

organisms [22]. According to previous reports, the action mechanism of SBPs is still

unknown and needs be clarified [22] but it is well accepted that SBPs are involved in

cancer prevention in human [9, 10, 26, 27] and in biotic and abiotic stress in plants [15,

19, 23, 42]. Some works consider that SBP, by sequestering Se in some conditions of

metal excess, may act as a detoxification molecule [24]. In the other hand, in human,

SBP together with Se – which in known to be an anti-proliferative molecule [43] – acted

as tumor suppressor, reducing proliferation of cancer cells [10, 44].

The sequence analysis of TcGPXs allowed the identification of the FPCNQF

domain containing a conserved cysteine residue (Fig. 5d). This motif was also identified

in several other plant and mammalian species [20]. Generally, the phylogenetic analysis

of TcGPXs with different GPX from plants and animals showed that they was very

conserved inclusive for each sub-group of GPX, i.e. PHGPXs vs GPXs (Fig. 3). The

sequence and structure conservation of TcGPXs also suggests a conservation of

function as indicated by other authors [45]. The molecular modeling of TcSBP and

34

TcGPXs followed by the docking of TcSBP-selenite and TcSBP-TcGPXs confirmed the

putative affinity of SBP with selenium compounds or macromolecules containing-

selenium [22, 41]. The physical and functional interaction between SBP and GPXs

already has been reported in human [9], but, to our knowledge, never has been

demonstrated in plants – even in silico. For this reason, our study is pioneer in studying

SBP-GPX interaction in plants and provides a first tentative of understanding the role of

these two molecules in plant-pathogen interaction. In human, correlations were made

between expression and/or activity of SBP and GPX [9, 10]. Briefly, the increase of

HsSBP expression was correlated to the inhibition of HsGPX activity, while de increase

of HsGPX expression was correlated to reduction of HsSBP expression [9, 10]. Here,

we analyzed the expression pattern of TcSBP, TcPHGPx and TcGPx2, and we observed

a negative correlation of gene expression between TcSBP and TcPHGPx, and between

TcSBP and TcGPx2 (Fig. 7). The general tendency observed was that, when TcSBP was

more expressed, TcGPx2 is less expressed or repressed (Catongo; Fig. 7b), and vice-

versa (TSH1188; Fig 7c). In Catongo, the TcSBP gene was more expressed than the

TcGPXs genes – mainly TcGPx2 – at 24 hai (asymptomatic phase) and then from 30 to

90 dai (Fig. 7a and b) that corresponds to the symptomatic phase involving PCD

process [35, 37]. This results could be related to the known SBP function, i.e. its

capacity to inhibit the cell proliferation, and consequently to its involvement in

apoptosis mechanism [10]. In rice, it has been shown that transgenic plants

overexpressing OsSBP acquired enhanced resistance to a virulent strain of the rice blast

biotrophic fungus [23]. In this case, OsSBP was involved in the accumulation of

reactive oxygen intermediates produced by NADPH oxidase, and the observed H2O2

accumulation was due to the reduction of scavenging enzyme activity [23]. In the cacao-

M. perniciosa interaction, which involved a hemibiotrophic fungus (i.e. having a

35

necrotrophic phase), TcSBP seemed to be involved in the last steps of the disease (from

30 dai), when the PCD and reactive oxygen species (ROS) production occurred and

favored the fungus phase transition from biotrophic to necrotrophic [35, 37, 46]. In

TSH1188, TcGPx2 was highly expressed from 24 to 72 hai, and significantly more

expressed than TcSBP (Fig. 7c) corroborating expression pattern in the resistant plant

previously published [47]. The antioxidant GPX enzymes eliminate ROS and reduce

H2O2 [19, 42], and in human, are known to be inhibitors of apoptosis [48]. Other studies

showed that calcium oxalate (Oxa) production is involved in PCD and is responsible for

the increase of ROS in plant [35]. However, when ROS production was inhibited, the

apoptotic-like-cell death induced by Oxa does not occur [35]. This suggests that in the

resistant cacao genotype infected with M. perniciosa, TcGPx2 may play a role in

protecting cells from oxidative stress and in reducing the progression of diseases by

inhibiting apoptosis. The TcPHGPx gene expression in the variety Catongo increased at

15 dai suggesting that TcPHGPx probably acted in direct reduction of phospholipid

hydroperoxide at this stage [14]. But TcPHGPx was reduced during the establishment of

the witches' broom disease in the susceptible variety. The increase of the PHGPX gene

expression has been related with the protection of the cells in various stress conditions

such as pathogenic attack, salt treatment and mechanical stimulation. A previous paper

remarkably increase in the level of Oryza sativum PHGPx was observed following

H2O2 stimulation [14]. Repression of GPx with increasing SBP expression observed in

cacao-M.perniciosa photosystem and the physical interaction between these proteins

was previously observed in several studies involving cancer. The mechanism by which

SBP1 and GPx1 regulate each other’s levels remains to be determined (Fang et al.,

2010; Zhang et al., 2013). One possible mechanism of inverse regulation might involve

competition for available of selenium in the cell (Fang et al., 2010).

36

Conclusion

In conclusion, here we obtained first report of physical interaction of SBP and GPX in

plant species. The knowledge of 3-D structure of the TcSBP, TcPHGPx and TcGPx2

was important for identifying the possible biochemical function. The data obtained in

the in silico analysis and by RT-qPCR indicate a putative functional interaction between

TcSBP and TcGPXs. Furthermore, such genes are differentially expressed during cacao-

M. perniciosa interaction. But the TcSBP gene it is not indicated for the induction of

resistance by overexpression. This because programmed cell death is a mechanism by

which M. perniciosa changes to dikaryotic phase. Thus overexpression of SBP may

exacerbate the severity of the symptoms of witches' broom disease. However use of

genes that inhibit apoptosis, e.g. TcGPXs may be a good candidate for obtaining cacao

plants resistant to M. perniciosa. The overexpression of TcGPx in TSH1188 in the

initial phase of witches' broom disease is an event which requires further examination

for a better understanding of the signaling pathways of cacao resistance to witches’

broom disease. The results shown here suggest then that these genes are important

targets for the management of witches' broom disease.

Methods

In silico analysis of TcSBP and TcGPXs

The TcSBP cDNA was identified from a library of T. cacao meristem (genotype

TSH1188) infected by M. perniciosa ([29]). The TcGPX sequences (TcPHGPx,

TcGPx2, TcGPx4, TcGPx6 and TcGPx8) were identified by a keyword search followed

by a blastp analysis on the CocoaGenDB database (http://cocoagendb.cirad.fr/cgi-

bin/gbrowse/theobroma/) [30]) using the GPX1 sequence from Homo sapiens

(accession number AAH70258) as query. The complete sequences of all the genes and

37

corresponding proteins were obtained from the CocoaGenDB database. Open reading

frame (ORF) detection was performed using the ORFinder software

(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Sequence homology search was made

with BLAST [49] on the National Center for Biotechnology Information (NCBI).

Multiple sequence alignment was performed with the ClustalW2 software [50]. The

prediction of theoretical isoelectric point (pI) and molecular weight (MW) was obtained

using the Expasy Molecular Biology Server (www.expasy.org). The conserved domain

and family protein were analyzed using the Pfam program

(http://pfam.sanger.ac.uk/search/sequence). Post-translational events were predicted

using the NetPhos 2.0 Server to identify putative sites of phosphorylation (Ser/Thr/Tyr)

[51] and NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) to identify

putative sites of N-glycosylation. Predictions of subcellular localization were conducted

with the programs TargetP [52] and PSORT [53]. Signal peptide presence was analyzed

using the SignalP 4.0 Server [54]. The TcSBP and TcGPXs were used, together with the

sequences of the homolog proteins from other plants and mammals, to construct an

unrooted phylogenetic tree by the neighbour joining method with the ClustalW2 [50]

and MEGA 6 [55] programs. For the phylogenetic analysis, only complete sequences

also having high identity were considered. The tree was constructed using the

neighbour-joining method of clustalW, with 1000 bootstraps.

Molecular modeling and docking analysis

The prediction of the three-dimensional (3-D) models of the TcSBP, TcGPx and

TcPHGPx proteins was obtained using the Swiss-Model server and the Automated

Protein Homology-modeling, which relies on the high similarity of target-template [56].

The crystal structure of the Sulfolobus tokodaii SBP was used as template (Protein Data

38

Bank code: 2ECE) to build the structural model of TcSBP while the crystal structure of

poplar (Populus trichocarpa) glutathione peroxidase (PtGPX5; Protein Data Bank code:

2p5q) was used as the template to build the 3-D models of TcPHGPx and TcGPx2. The

stereochemical quality of the models was evaluated using the Procheck [57] and Anolea

programs [58]. The 3-D model visualization was obtained using the PyMol (The

PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.) and

Discovery Studio 4.0. For docking analysis of TcSBP 3-D model (receptor) with

inorganic selenium compounds (ligands), selenite and selenite were search by keyword

in the PubChem databases (http://pubchem.ncbi.nlm.nih.gov). The 2D structure of these

compounds were copied in Similes and saved in PDB format using the Marvin program,

and then used for molecular docking. The ligand and receptor molecules were prepared

using the AutoDockTools 1.5.6 [59]. The grid definition, adjusted to active site, was set

up manually by following the recommendations of the program manual [59, 60]. The

ligand and receptor structures were then saved in pqbqt format to be used for docking

calculations. AutoDock Vina was used to perform Docking Scoring for each ligand-

receptor complex [60]. Before running each Docking calculation, a configuration file

was generated with information about grid size and coordinates and indicating the

ligand and receptor files. The reports for each calculation were analyzed to obtain

affinity energy (kCal/Mol) values for each ligand conformation in its respective

complex. PyMol program was used to verify the ideal complex by considering all

stereochemical aspects previously evaluated and the free-energy results, and then the

best ligand i.e., that fit best in the active site, was selected. The molecular docking

protein-protein was performed in ROSIE (http://rosie.rosettacommons.org/about), a

molecular modeling software package that provides experimentally tested and rapidly

39

evolving tools for the 3D structure prediction, as well as high-resolution design of

proteins, nucleic acids, and non-natural polymers [61].

Plant material

Plant material was obtained as previously described [62]. Seeds of T. cacao genotypes

Catongo (susceptible to M. perniciosa) and TSH1188 (resistant to M. perniciosa) were

germinated and grown at CEPLAC/CEPEC (Ilhéus, Bahia, Brazil) greenhouses. Twenty

to thirty days after germination, the apical meristems of the plantlets were inoculated by

the droplet method [63] with a basidiospore suspension of M. perniciosa (inoculum

from isolate 4145 maintained in the CEPLAC/CEPEC phytopathological M. perniciosa

collection under number 921 of the WFCC;

http://www.wfcc.info/index.php/collections/display). After inoculation, the plantlets

were kept for 24 h at 25 ± 2°C and 100% humidity. Rate of disease fixation based on

presence/absence of symptoms [64] in each genotype, was evaluated 60 days after

inoculation (dai); disease rate was 45% and 80% for TSH1188 and Catongo,

respectively. Moreover, the presence of M. perniciosa in the plant material was checked

by semi-quantitative RT-PCR using specific M. perniciosa actin primers [65]; both

genotypes presented fungus incidence (data not shown) coherent with previous data

obtained in the same conditions of plant culture and inoculation [36]. Apical meristems

were harvested at 24, 48 and 72 hours after inoculation (hai), and 8, 15, 30, 45, 60 and

90 dai. Non-inoculated plants (controls) were kept and harvested under the same

conditions at 24 and 72 hai, and 30, 60 dai and 90 dai. For each genotype and at each

harvesting time (for inoculated and non inoculated plants), 20 samples were collected (1

sample = 1 apical meristem of 1 cacao plantlet). The 20 samples collected from one

genotype at one harvesting time were pooled; thus 9 inoculated and 5 non inoculated

40

(control) samples were immediately frozen in liquid nitrogen and stored at -80°C until

use. Pooling samples before RNA extraction has the advantage of reducing the variation

caused by biological replication and sample handling [66].

Reverse transcription quantitative PCR analysis

Total RNA was extracted from macerated samples using the RNAqueous Kit®

(Ambion) according to the manufacturer’s instructions, with modifications as

previously described [62]. The synthesis of the first cDNA strand was carried out using

Revertaid Fisrt Strand cDNA Synthesis Kit according to the manufacturer’s instructions

(Thermo Scientific). The cDNA quantification was carried out on the GeneQuant pro

UV/Vis spectrophotometer (Amersham). For the qPCR analysis, two cacao endogenous

reference genes were used: the malate dehydrogenase (MDH) and β-actin (ACT),

previously identified as T. cacao housekeeping genes [67] and tested in our

experimental conditions (same plant material, same equipment [62]). Specific primers

and amplified regions containing different size, melting temperature, GC content and

GC/AT ratio were defined to avoid cross-reaction between genes from cacao SBP and

GPX family (Supplementary material 7). The expression analysis of TcSBP, TcPHGPx

and TcGPx2 was performed using standard settings of the ABI PRISM 7500 and

Sequence Detection System (SDS) software, version 1.6.3 (Applied Biosystems). The

qPCR reaction consisted of 10 ng/μl of cDNA, 1 μM of each primer from reference or

targets genes (Supplementary material 7) and 11 μl of Power SYBR Green Master Mix

(Applied Biosystems) in a total volume of 22 μl. Cycling conditions were: 50°C for 2

min then 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30s and

60°C for 1 min. To verify that each primer pair produced only a single PCR product, a

dissociation analysis was carried out from 60°C to 95°C and analyzed with the

41

Dissociation Curve 1.0 program (Applied Biosystems). The expression analysis of

TcSBP, TcPHGPx and TcGPx2 in meristems of cacao plantlets inoculated or not with

M. perniciosa, was obtained by RT-qPCR using 5 experimental replicates. The relative

expression was analyzed with the comparative Ct method (2-ΔΔCt) using MDH and ACT

as endogenous reference genes and non-inoculated plants (control) as calibrator.

Statistical analysis was made using the SASM-Agri software which tested the

experiments as a completely randomized design. t-test and F-test (ANOVA) were

applied with a critical value of 0.01. The Scott-Knott (P≤0.01) test was employed for

mean separation when F-values were significant.

Availability of supporting data

The data sets supporting the results of this article are included within the article and its

additional files.

Abbreviations

ACT: actin; dai: days after inoculation; GPX: glutathione peroxidase; GSH: reduced

glutathione; hai: hour after inoculation; pI: isoeletric point; MDH: malate

dehydrogenase; MW: molecular weight; ORF: open reading frame; PCD: programmed

cell death; PHGPX: phospholipid hydroperoxide glutathione peroxidase; S: sulfur; SBP:

selenium-binding protein; Se: selenium; SeMet: selenomethionine; Secys:

selenocysteine; UTR: untranslated regions.

Competing interest

No conflicts of interest to declare.

Authors’ contribution

42

AMMA was responsible for the execution of the all the experimental steps; AMMA,

SPM, BSA and FM analyzed and discussed the data; AMMA and FM wrote the

manuscript; SPM and EML gave support in qPCR experiment; BSA gave support in

modeling and docking analyses; KPG was responsible for plant material production and

inoculation with M. perniciosa; FM and BSA were responsible for the conception and

design of the experiments; FM was responsible for the financial support of the research

and for the advising of AMMA, EML and SPM.

Acknowledgements

The work of AMMA was supported by the Fundação de Amparo à Pesquisa do Estado

da Bahia (FAPESB). The work of SPM and EML was supported by the Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This research was supported

by the FAPESB project (DTE0038/2013) coordinated by FM. The authors thank

Francisca Feitosa Jucá (UESC/Ceplac) and Louise Araújo Sousa (Ceplac) for technical

help in plant inoculation experiments, Dr. Gesilvado Santos (UESB) for advices and

discussion about SBP role.

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47

Tables

Table 1. Gene and protein characteristics of SBP and GPX family from T. cacao. nd: non determined.

Name

Gene data Protein data

Identification* Sequence

start*

Sequence

end*

ORF size

(bp)

Size

(aa)

Molecula

weight (Da) pI

Subcellular

localization Phosphorylation sites Glycosylation sites

TcSBP Tc04_p020340 17507342 17511657 1431 476

52732.7

5.74

nd

S23 S56 S91 S186 S206 S288 S290 S296 S320

S411 T17 T39 T49 T201 T284 T435 Y15 Y59

Y108 Y227 Y404 -

TcPHGPx Tc09_p001340 728482 730367 720 239

26477.2

9.08

Chloroplast S33 S36 S40 S43 S44 S58 S70 S96 S98 S122

S153 T20 T78 T81 N120

TcGPx2 Tc05_p000210 101857 104202 789 262

29708.3

9.11

Secretory S31 S42 S57 S59 S62 S152 T47 T185 T246 Y44

Y92 Y147 N165 N227

TcGPx4 Tc01_p028750 24070592 24074869 516 171

19175.9

9.14

nd S11 S28 S121 T16 T83 Y52 Y116 N50 N122 N134

TcGPx6 Tc03_p027320 23147873 23149733 780 235

25973.7

8.93

Chloroplast

S25 S29 S31 S32 S43 S52 S57 S63 S74 S75 S78

S95 S188 S189 S197 S225 T83 T221 Y118 Y128

Y166 Y183

N12 N117 N201

TcGPx8 Tc03_p027330 23150262 23153108 573 190 21600.4

5.27

nd S5 S11 S28 S50 S159 S173 S176 T16 T54 Y13

Y69 Y79 Y134 N68

* Gene identification and position as indicated in CocoaGenDB database

48

Figure legends

Figure 1. Structural features of SBP gene and glutathione peroxidase gene family of T. cacao.

Untranslated regions (UTRs) are indicated by black squares, exons by gray squares, and

introns by black lines. The lengths of the UTR, exons and introns are drawn to scale.

Figure 2. Phylogenetic analysis, using amino acid data, of TcSBP and SBP from other

species. The green and red boxes indicate plant and mammalian SBPs, respectively. The

amino acids sequences were derived from the GenBank under the following accession

numbers: VvSBP, Vitis vinífera (XP_002267004.1); CsSBP, Citrus sinensis (XP_006492530.1);

StSBP, Solanum tuberosum (XP_006360921.1); PtSBP, Populus trichocarpa (XP_006373096.1);

RcSBP, Ricinus communis (XP_002520613.1); TcSBP, Theobroma

cacao (XP_007034202.1); CsSBP, Cucumis sativus (XP_004138463.1); MsSBP, Medicago

sativa (CAC67501.1); GmSBP, Glycine max (KHN24031.1); PvSBP, Phaseolus

vulgaris (XP_007163807.1); ShSBP, Sarcophilus harrisii (XP_003773388.1); MmSBP, Mus

musculus (AAA40104.1); RnSBP, Rattus norvegicus (NP_543168.1); HgSBP, Heterocephalus

glaber (XP_004875930.1); LaSBP, Loxodonta africana (XP_010587740.1); BmSBP,

Bos mutus (XP_005895013.1); TtSBP, Tursiops truncatus (XP_004328520.1); PtSBP, Pan

troglodytes (XP_001172033.2); HsSBP, Homo sapiens (AAB02395.1); GggSBP, Gorilla gorilla

gorila (XP_004026682.1).

Figure 3. Phylogenetic relationship between the amino acid sequences of TcGPXs and others plants

and mammalian GPXs. Only complete sequences were considered. The tree was constructed using the

neighbour-joining method of clustalW, with 1000 bootstraps. The box green indicates the plants GPXs

and box red indicates the mammalians GPXs. The amino acids sequences were derived from the

GenBank under the following accession numbers: TcPHGPx, Theobroma cacao

(XP_007011699.1); GaPHGPx, Gossypium arboreum (KHG09053.1); AtPHGPx, Arabidopsis

thaliana (NP_180080.1); VvGPx2, Vitis vinifera (XP_002263327.1); TcGPx2, Theobroma cacao

(XP_007026518.1); PeGPx2, Populus euphratica (XP_011026385.1); GmGPx4, Glycine

max (NP_001238132.1); TcGPx4, Theobroma cacao (XP_007050669.1); PtGPx4, Populus

49

trichocarpa (XP_002320392.1); GmGPx6, Glycine max (KHN19874.1); TcGPx6, Theobroma

cacao (XP_007040204.1); FvGPx6, Fragaria vesca (XP_004298758.1); TcGPx8, Theobroma

cacao (XP_007040205.1); CsGPx8, Citrus sinensis (XP_006476628.1); SlGPx8, Solanum

lycopersicum (XP_004252596.1); MmGPX, Mus musculus (NP_032186.2); BtGPX, Bos

taurus (1GP1_A); EcGPX, Equus caballus (NP_001159951.1); HsGPX, Homo

sapiens (NP_000572.2); MmGPX, Macaca mulata (NP_001152770.1).

Figure 4. Tridimensional structure of TcSBP, TcPHGPx and TcGPx2 obtained by homology

modeling with StSBP from Sulfolobus tokodaii (PDB code 2ECE.pdb) and PtGPx5 from Populus

trichocarpa (2P5R.pdb). (a) Alignment of TcSBP with StSBP. (b) Alignment of TcPHGx and TcGPx2

with PtGPx5. (c) Secondary structure of TcSBP. (d) Secondary structure of TcPHGPx. (e) Secondary

structure of TcGPx2.

Figure 5. The conserved motifs of TcSBP and TcGPx2. (a). The conserved motifs DELHH and HGD

of TcSBP are indicated in orange and pink, respectively. (b). Focus on the conserved motifs of TcSBP.

(c). The conserved motif FPCNQF of TcGPx2 is indicated in pink. (d). Focus on the conserved motifs

of TcSBP. The conserved cysteine residue of TcGPx2 is highlighted in yellow.

Figure 6. Docking between TcSBP and selenite, TcSBP and TcGPx2, and TcSBP and

TcPHGPX. (a). Structure of SBP showing the conserved CSSS motif (in pink). (b) Focus on

the CSSC motif from TcSBP. (c) Interaction of selenite with the CSSC motif of TcSBP. (d)

Interaction between TcSBP (in green) and TcGPx2 (in blue) which occurred near to the CSSC

motif (in pink). (e) Interaction between TcSBP (in green) and TcPHGPX (in yellow-green)

distant from the CSSC motif (in pink).

Figure 7. Relative expression of TcSBP, TcPHGPx and TcGPx2 in cacao meristems

inoculated or not (control) with M. perniciosa. (a). Representation of the plant symptoms and

fungus phase during the infection time course in Catongo genotype. The harvesting times of

inoculated plants are indicated on the top of the figure. (*) indicates the times that were

harvested also in the non-inoculated (control) plants. (b) Relative expression observed in

Catongo. (c). Relative expression observed in TSH1188. Different letters indicate significant

50

statistical difference between samples by the Scott-Knott test p(p≤0.01). t-test were applied

with a critical value of 0.01: upper case letters correspond to statistics between each of the

genes on different harvesting times for each genotype while lower case letters correspond to

statistics between the three genes for each harvesting time. hai: hours after inoculation; dai:

days after inoculation.

51

Figures

Figure 1. Alves et al.

52

Figure 2. Alves et al.

53

Figure 3. Alves et al.

54

Figure 4. Alves et al.

55

Figure 5. Alves et al.

56

Figure 6. Alves et al.

57

Figure 7. Alves et al.

58

Supplementary materials

Supplementary material 1. Alignment of the amino acid sequence of TcSBP with SBPs from other

plants species and from human. The sequences used for alignment are: PtSBP, Populus

trichocarpa (XP_006373096.1); GmSBP, Glycine max (KHN24031.1); CsSBP, Cucumis

sativus (XP_004138463.1); TcSBP, Theobroma cacao (XP_007034202.1); HsSBP, Homo

sapiens (AAB02395.1). Gaps introduced to get the best alignment are indicated by (-), (*) represents

identical amino acids between all sequences, (.) and (:) represent conserved substitutions and semi-

conserved substitutions, respectively. The conserved motifs DELHH, CSSC and HGD are highlighted

in blue, red and black, respectively.

TcSBP1_Cacao ---------------------------------------MAGNGTGCCKTGPGYATPLEA

PtSBP_Poplar --------------------------------MATGTEVVSDNGHGCCKKGPGYATPLEA

CsSBP_Cucumber ----------------------------MATDTAVAEHRSSNNGHGCCAKGPGYATPLDA

GmSBP1_Soybean -----------------------------------MSSVLEHGVVGCCKSGPGYATPLEA

HsSBP1_Human MRLEWGPRPAALPWPAGMCAAERAEGAFTLQSVAQPMRPIASTATKCGNCGPGYSTPLEA

* ****:***:*

TcSBP1_Cacao MSGPREALIYVTCVYTGTGREKPDFLATVDVDPNSPTYSKVIHRLPVPYLGDELHHSGWN

PtSBP_Poplar MSGPRESLLYVTCVYSGTGIEKPDYLATVDVDPNSPTYSKVIHRLPMPNVGDELHHTGWN

CsSBP_Cucumber MSGPREKLLYVTCLYTGTGREKPDYLATVDADPSSSSYSKVIHRLPVPYIGDELHHSGWN

GmSBP1_Soybean MSGPRESLIYVTAVYTGTGIEKPDYLATVDIDPNSPTYSKVIHRLRVPYLGDELHHTGWN

HsSBP1_Human MKGPREEIVYLPCIYRNTGTEAPDYLATVDVDPKSPQYCQVIHRLPMPNLKDELHHSGWN

*.**** ::*:..:* .** * **:***** **.*. *.:***** :* : *****:***

Tc1SBP1_Cacao SCSSCHGDPSAERRFLILPSLVSGHIYVIDTQTNPKAPSLHKVVDPEDIVQKTGLAYPHT

Pt1SBP_Poplar SCSSCHGDPSAARRYLVLPSLISGRIYAIDTLKDPRAPSLHKVVEPADIVNKTGLAYPHT

CsSBP_Cucumber SCSSCYGDSSAQRRFLVLPSLVSGRIYIVDTQKNPRAPSLHKVVEPADIVQKTGLSYPHT

GmSBP1_Soybean SCSSCHGDPSADRRFLIAPALVSGRIYVVDVKTNPRAPSLHKVVEPADIIQKTGLAYPHT

HsSBP1_Human TCSSCFGDSTKSRTKLVLPSLISSRIYVVDVGSEPRAPKLHKVIEPKDIHAKCELAFLHT

:****.**.: * *: *:*:*.:** :*. .:*:**.****::* ** * *:: **

TcSBP1_Cacao SHCLASGDIMVSCLGDKDGNAKGNGFLLLDSEFNVKGRWEKPGHSPLFGYDFWYQPRHKT

PtSBP_Poplar SHCLASGDVMVSCLGDKDGNAEGNGFLLLDSEFNVKGRWEKPGHSPTFGYDFWYQPRHNI

CsSBP_Cucumber AHCLASGDILVSCLGDKDGNAEGNGFLLLDSEFNVKGRWEKPGHSPAFGYDFWYQPRHKT

GmSBP1_Soybean SHCLASGDIMISCLGDKDGNAAGNGFLLLDSEFNVKGRWEKPGHSPLFGYDFWYQPRHNT

HsSBP1_Human SHCLASGEVMISSLGDVKGNGKGGFVLLDGETFEVKGTWERPGGAAPLGYDFWYQPRHNV

:******::::*.*** .**. *. .** .. *:*** **:** :. :**********:

TcSBP1_Cacao MISSSWGAPAAFTKGFNLQHVADGLYGRHLYVYSWPDGELKQTLDLGDSGLLPLEIRFLH

PtSBP_Poplar MISSSWGAPAAFTKGFNLQHVADGLYGRHLNVYSWPNGELKQTLDLGDTGLLPLEIRFLH

CsSBP_Cucumber MISSSWGAPLAFTKGFNLQHVSDGLYGRHLFVYSWPDGELKQTLDLGNTGLIPLEIRFLH

GmSBP1_Soybean MISTSWGAPSAFTKGFNLQHLSDGLYGRHLHVYSWPGGELRQTLDLGDSGLLPLEIRFLH

HsSBP1_Human MISTEWAAPNVLRDGFNPADVEAGLYGSHLYVWDWQRHEIVQTLSLKDG-LIPLEIRFLH

***:.*.** .: .*** .: **** ** *:.* *: ***.* : *:********

TcSBP1_Cacao DPSKDTGFVGCALTSNMVRFFKTKDGSWSHEVAISVKPLKVQNWILPEMPGLITDFLISL

PtSBP_Poplar DPSKDSGFVGCALTSNMVRFFKTPDGSWSHEVAISVKPLKVQNWILPEMPGLVTDFLISL

CsSBP_Cucumber DPSKDIGFVGCALASTMVRFFKTQDGSWNHEVAISVKSLKVQNWILPEMPGLITDFLISL

GmSBP1_Soybean DPAKDTGFVGSALTSNMIRFFKTQDESWSHEVAISVKPLKVQNWILPEMPGLITDFLISL

HsSBP1_Human NPDAAQGFVGCALSSTIQRFYKNEGGTWSVEKVIQVPPKKVKGWLLPEMPGLITDILLSL

:* ****.**:*.: **:*. . :*. * .*.* . **:.*:*******:**:*:**

TcSBP1_Cacao DDRFLYFANWLHGDVRQYNIEDPKNPVLAGQVWVGGLIQNGSPVVAVIEDGKTWQCNVPE

PtSBP_Poplar DDRFLYFVNWLHGDVRQYSIEDPEKPVLKGQVWVGGLIQKGSSVVAEGEDGKTWQYDVPE

CsSBP_Cucumber DDRFLYFSNWLHGDIRQYNIEDPKNPVLTGQVWVGGLFQKGSPVVAVTDDGQPYQSDVPS

GmSBP1_Soybean DDRFLYFVNWLHGDIRQYNIENLKNPKLTGQVWVGGLIQKGSPVVAITDDGETWQAEVPE

HsSBP1_Human DDRFLYFSNWLHGDLRQYDISDPQRPRLTGQLFLGGSIVKGGPVQVLEDEELKSQPEPLV

******* ******:***.*.: :.* * **:::** : :*..* . :: * :

TcSBP1_Cacao IQGHRLRGGPQMIQLSLDGKRLYVTNSLFSTWDRQFYPELVEKGSHMLQIDVDTEKGGLK

PtSBP_Poplar IQGHRLRGGPQMIQLSLDGKRLYVTNSLFSTWDRQFYPELMEKGSHMLQIDVDTEKGGLA

CsSBP_Cucumber VQGHRLRGGPQMIQLSLDGKRLYVTNSLFSAWDCQFYPELKEKGSHMLQIDVNSEKGGMA

GmSBP1_Soybean IQGNKLRGGPQMIQLSLDGKRLYATNSLFSTWDKQFYPELVQKGSHIIQIDVDTEKGGLK

HsSBP1_Human VKGKRVAGGPQMIQLSLDGKRLYITTSLYSAWDKQFYPDLIREGSVMLQVDVDTVKGGLK

::*::: **************** *.**:*:** ****:* .:** ::*:**:: ***:

TcSBP1_Cacao VNPYFFVDFGAEPDGPSLAHEMRYPGGDCTSDIWI

PtSBP_Poplar INPNYFVDFAAEPDGPSLAHEMRYPGGDCTSDIWI

CsSBP_Cucumber INPNFFVDFEAEPDGPALAHEMRYPGGDCTSDIWI

GmSBP1_Soybean INPNFFVDFGAEPDGPSLAHEMRYPGGDCTSDIWI

HsSBP1_Human LNPNFLVDFGKEPLGPALAHELRYPGGDCSSDIWI

:** ::*** ** **:****:*******:*****

59

Supplementary material 2. Alignment of the amino acid sequence of TcGPXs with the GPX from

human (HsGPX). Gaps introduced to get the best alignment are indicated by (-), (*) represents

identical amino acids between all sequences, (.) and (:) represent conserved substitutions and semi-

conserved substitutions, respectively. The conserved motif FPCNQF is highlighted in blue, and inside

it, the conserved cysteine residue is indicated in red.

TcGPX4 ------------------------------------------------------------

TcGPX6 -MLCSPTYLFRRNLSAVAVSASLLLSKRLSPSSKQTLLSFPQISPVSLVSP--SIETGFS

TcPHGPx MASMPFSATFPSYLHDLSQTKKIPVMPSSWPFSIPSIESSLGSSKSGFLQHGFSLQSSSV

TcGPX2 ---------------------------------------MMHWLRFTNLVSLVFLGFAFF

TcGPX8 ------------------------------------------------------------

HsGPX1 ------------------------------------------------------------

TcGPX4 ----------MGASESVPQKSIHQFTVKDNKGQD-VDLSIYEGKVLLVVNVAS-------

TcGPX6 RSFLGSLRFDHIMAGQSSKGSIHDFTVKDARGND-VDLSIYKGKVLLIVNVAS-------

TcPHGPx PGFVFKSRSSGIYARAATEKTLYDYTVKDIDGKD-VSLSRFKGKVLLIVNVAS-------

TcGPX2 LYFHIYPSSSHQNMAENAPKSVYEFTVKDIRGND-VSLSEYSGKVLLIVNVAS-------

TcGPX8 ----------MASQSTKNPESIYDFTVKDAKGNV-VDLSAYKGKVLLIVNVASKWYLRSD

HsGPX1 ------MCAARLAAAAAAAQSVYAFSARPLAGGEPVSLGSLRGKVLLIENVASLU-----

::: ::.: * *.*. *****: ****

TcGPX4 -----------KCGLTDSNYTQLTDLYSKYKDQGLEILAFPCNQFLKQEPGTEQEVQQFA

TcGPX6 -----------QCGLTNSNYTELSQLYEKYKDQGLEILAFPCNQFGGQEPGNNEQILEFA

TcPHGPx -----------KCGLTTSNYSELSHIYEKYKTQGFEILAFPCNQFGGQEPGSNPEIKQFA

TcGPX2 -----------KCGLTHSNYKELNVLYEKYKNQGFEILAFPCNQFAGQEPGTNEHIQEVA

TcGPX8 SVINTGRLFFATDGMTNPNYTELNQLYEKYKDQGLEILAFPCNQFGEEEPGSNDQIAVFV

HsGPX1 -------------GTTVRDYTQMNELQRRLGPRGLVVLGFPCNQFGHQENAKNEEILNSL

* * :*.::. : : :*: :*.****** :* ..: .:

TcGPX4 CTR-----YKAEYPIFRKVRVNGPKTEPVYKFLKSNKSG-------------------FL

TcGPX6 CTR-----FKAEYPIFDKVDVNGEKTAPIYKFLKSSKGG-------------------LF

TcPHGPx CTR-----FKAEFPIFDKVDVNGPNTAPVYQFLKSNAGG-------------------FL

TcGPX2 CTM-----FKAEFPIFDKVEVNGKNSAPLYKFLKSVKGG-------------------YF

TcGPX8 CTR-----FRSEFPIFDKIEVNGDNASPLYKYLKLGKWG-------------------IF

HsGPX1 KYVRPGGGFEPNFMLFEKCEVNGAGAHPLFAFLREALPAPSDDATALMTDPKLITWSPVC

:..:: :* * *** : *:: :*: .

TcGPX4 GSRIKWNFTKFLVDKNGHVLGRYGPTTAPLAIEADIKKALGVDT----------------

TcGPX6 GDSIKWNFSKFLVDKEGNVVDRYAPTTSPLSIEKDIKKLLA-------------------

TcPHGPx GDLVKWNFEKFLVDKNGKVVERYPPTISPFQIEKDIQKLLAA------------------

TcGPX2 GDAIKWNFTKFLVDKEGKVVERYAPTTSPLKIEQRTRDIAWNFQGVHGSKAWLVFGGLRL

TcGPX8 GDDIQWNFAKFLVSKDGQVVHRYYPTTSPLSLEYDIKKLLGLGQE---------------

HsGPX1 RNDVAWNFEKFLVGPDGVPLRRYSRRFQTIDIEPDIEALLSQGPSCA-------------

. : *** ****. :* : ** .: :* .

TcGPX4 --------------------------------------------

TcGPX6 --------------------------------------------

TcPHGPx --------------------------------------------

TcGPX2 VVFISLLSNVSGWMITSSTMNYLCGAFTHRVGCKASHKIICNNL

TcGPX8 --------------------------------------------

HsGPX1 --------------------------------------------

60

Supplementary material 3. Alignment of amino acid sequence of TcSBP with others plants and

mammalians SBPs. The sequences used for alignment are: VvSBP, Vitis

vinífera (XP_002267004.1); CsSBP, Citrus sinensis (XP_006492530.1); StSBP, Solanum

tuberosum (XP_006360921.1); PtSBP, Populus trichocarpa (XP_006373096.1); RcSBP, Ricinus

communis (XP_002520613.1); TcSBP, Theobroma cacao (XP_007034202.1); CsSBP, Cucumis

sativus (XP_004138463.1); MsSBP, Medicago sativa (CAC67501.1); GmSBP, Glycine

max (KHN24031.1); PvSBP, Phaseolus vulgaris (XP_007163807.1); ShSBP, Sarcophilus

harrisii (XP_003773388.1); MmSBP, Mus musculus (AAA40104.1); RnSBP, Rattus

norvegicus (NP_543168.1); HgSBP, Heterocephalus glaber (XP_004875930.1); LaSBP, Loxodonta

africana (XP_010587740.1); BmSBP, Bos mutus (XP_005895013.1); TtSBP, Tursiops

truncatus (XP_004328520.1); PtSBP, Pan troglodytes (XP_001172033.2); HsSBP, Homo sapiens

(AAB02395.1); GggSBP, Gorilla gorilla gorila (XP_004026682.1). Gaps introduced to get the best

alignment are indicated by (-), (*) represents identical amino acids between all sequences, (.) and (:)

represent conserved substitutions and semi-conserved substitutions, respectively.

GmSBP1_Soybean -----------------------------------MSSVLEHGVVGCCKS 15

PvSBP_Bean -----------------------------------MS-----NGHGCCKT 10

MsSBP_Alfalfa ---------------------------MGTVLQHAVVSEKVNNQQGCCKS 23

Tc1SBP1_Cacao ----------------------------------MAG-----NGTGCCKT 11

Cs1SBP2_Cucumber ----------------------------------MADGTSNSNGNACCRH 16

Pt1SBP_Poplar --------------------------------MATGTEVVSDNGHGCCKK 18

RcSBP_Castor_bean --------------------------------------MVKDS--CCMNK 10

VvSBP1_Wine_grape ------------------------------------------MEIGCCKK 8

Cs2SBP2S_Weet_orange ----------------------------MATDTAVAEHRSSNNGHGCCAK 22

StSBP1_Potato ----------------------------MATDMEVLQNGKAAAVNGCCKK 22

BmSBP1_Yak --------------MGGLGGAACKEGAFALQSVGQPTSPIVSTATKCGKC 36

TtSBP1_Dolphin ------------------------------------------MATKCGKC 8

HsSBP1_Human MRLEWGPRPAALPWPAGMCAAERAEGAFTLQSVAQPMRPIASTATKCGNC 50

Pt2SBP1_Chimpanzee MRLEWGPRPAALPWPAGMCAAGRAEGAFTLQSVAQPMRPIASTATKCGNC 50

GggSBP1_Western_lowland_gorila ------------------------------------------MATKCGNC 8

LaSBP1_Elephant ------------------------------MTIRNSLQEDAFFDTKCRKC 20

MmSBP1_House_mouse ------------------------------------------MATKCTKC 8

RnSBP1_Norway_rat ------------------------------------------MATKCTKC 8

HgSBP1_Naked_mole-rat ------------------------------------------MATKC-KC 7

ShSBP1_Tasmanian_devil -------------------------------------------MAKCEKC 7

*

GmSBP1_Soybean GPGYATPLEAMSGPRESLIYVTAVYTGTGIEKPDYLATVDIDPNSPTYSK 65

PvSBP_Bean GPGYASPLEAMAGPRESLIYVTAVYSGTGIEKPDYVATVDVDPNSATFSK 60

MsSBP_Alfalfa GPGYASPLEAMSGPRETLIYVTAVYAGTGIEKPDYLATVDLDPNSPTYSK 73

Tc1SBP1_Cacao GPGYATPLEAMSGPREALIYVTCVYTGTGREKPDFLATVDVDPNSPTYSK 61

Cs1SBP2_Cucumber GPGYATPLEAMSGPRETLIYVTAVYSGTGINKPDYLATVDVDPNSPNYSK 66

Pt1SBP_Poplar GPGYATPLEAMSGPRESLLYVTCVYSGTGIEKPDYLATVDVDPNSPTYSK 68

RcSBP_Castor_bean GPGYATPLEAMSGPRESLIYVTCVYSGTGIDKPDYLATVDINPNSPTYSQ 60

VvSBP1_Wine_grape GPGYATPLEAMSGPRESLLYVTCIYTGSGKGKPDYLATVDVDPSSPSYSK 58

Cs2SBP2S_Weet_orange GPGYATPLDAMSGPREKLLYVTCLYTGTGREKPDYLATVDADPSSSSYSK 72

StSBP1_Potato GPGYASPLAAMDGPKESLIYVTCIYTGMGRGKPDYLATVDVDPKSPSYSK 72

BmSBP1_Yak GPGYPSPLEAMKGPREELVYLPCIYRNTGTEAPDYLATVDVNPKSPQYSQ 86

TtSBP1_Dolphin GPGYPSPLEAMKGPREEIVYLPCIYRNTNTEAPDYLATVDVDPKSPQYCQ 58

HsSBP1_Human GPGYSTPLEAMKGPREEIVYLPCIYRNTGTEAPDYLATVDVDPKSPQYCQ 100

Pt2SBP1_Chimpanzee GPGYSTPLEAMKGPREEIVYLPCIYRNTGTEAPDYLATVDVDPKSPQYCQ 100

GggSBP1_Western_lowland_gorila GPGYSTPLEAMKGPREEIVYLPCIYRNTGTEAPDYLATVDVDPKSPQYCQ 58

LaSBP1_Elephant GPGYPTPLEAMKGPREELVYLPCIYRNTGIEAPDYLATVDVDPKSPHYSQ 70

MmSBP1_House_mouse GPGYSTPLEAMKGPREEIVYLPCIYRNTGTEAPDYLATVDVDPKSPQYSQ 58

RnSBP1_Norway_rat GPGYATPLEAMKGPREEIVYLPCIYRNTGIEAPDYLATVDVDPKSPHYSQ 58

HgSBP1_Naked_mole-rat GPGYPSPREAMKGPREEIIYLPCIYRNTGTEAPDYLATVDIDPNSPQYCQ 57

ShSBP1_Tasmanian_devil GPGYPTPLDAMKGPREELLYLPCIYRNTGTEAPDYLATIDVDPKSPSYSQ 57

****.:* ** **:* ::*:..:* . . **::**:* :*.*. :.:

GmSBP1_Soybean VIHRLRVPYLGDELHHTGWNSCSSCHGDPSADRRFLIAPALVSGRIYVVD 115

PvSBP_Bean VIHRLPVPYLGDELHHTGWNSCSSCFGDPSAQRRFLIVPALVSGRVYVID 110

MsSBP_Alfalfa VIHRLPVPYVGDELHHTGWNSCSSCHGDPSAQRRFLIVPGIVSGRVYVID 123

Tc1SBP1_Cacao VIHRLPVPYLGDELHHSGWNSCSSCHGDPSAERRFLILPSLVSGHIYVID 111

Cs1SBP2_Cucumber VIHRLSFPYLGDELHHSGWNSCSSCHGDPSADRRFLILPSLLSGRIYVID 116

Pt1SBP_Poplar VIHRLPMPNVGDELHHTGWNSCSSCHGDPSAARRYLVLPSLISGRIYAID 118

RcSBP_Castor_bean VIHRLPVPYVGDELHHSGWNACSSCHGDPSANRRFLILPSLISGRIYVID 110

VvSBP1_Wine_grape VIHRLPVPYLGDELHHSGWNSCSSCHGDSSQERRFLVLPSLVSGRIYAID 108

Cs2SBP2S_Weet_orange VIHRLPVPYIGDELHHSGWNSCSSCYGDSSAQRRFLVLPSLVSGRIYIVD 122

StSBP1_Potato VIHRLPMPYEGDELHHSGWNSCSSCYGDPSAARRYLVLPSLVSGRIYAID 122

BmSBP1_Yak VIHRLPMPNLKDELHHSGWNTCSSCFGDSTKSRTKLVLPSLISSRVYVVD 136

TtSBP1_Dolphin VIHRLPMPHLKDELHHSGWNTCSSCFGDSTKSRTKLLLPSLISSRIYVVD 108

61

HsSBP1_Human VIHRLPMPNLKDELHHSGWNTCSSCFGDSTKSRTKLVLPSLISSRIYVVD 150

Pt2SBP1_Chimpanzee VIHRLPMPNLKDELHHSGWNTCSSCFGDSTKSRTKLVLPSLISSRIYVVD 150

GggSBP1_Western_lowland_gorila VIHRLPMPNLKDELHHSGWNTCSSCFGDSTKSRTKLVLPSLISSRIYVVD 108

LaSBP1_Elephant VIHRLPMPNLKDELHHSGWNTCSSCFGDSTKTRTKLVLPSLISSRIYVVD 120

MmSBP1_House_mouse VIHRLPMPYLKDELHHSGWNTCSSCFGDSTKSRNKLILPGLISSRIYVVD 108

RnSBP1_Norway_rat VIHRLPMPHLKDELHHSGWNTCSSCFGDSTKSRDKLILPSIISSRIYVVD 108

HgSBP1_Naked_mole-rat VIHRLPMPYLKDELHHSGWNTCSSCFGDSTKSRTKLMLPCLISSRIYVVD 107

ShSBP1_Tasmanian_devil VIHRLPMPHLKDELHHSGWNTCSSCFGDSSKSRTKLILPSLISSRVYVVD 107

***** .* *****:***:****.**.: * *: * ::*.::* :*

GmSBP1_Soybean VKTNPRAPSLHKVVEPADIIQKTGLAYPHTSHCLASGDIMISCLGDKDGN 165

PvSBP_Bean VKTDPKAPSLHKVVEPADIIQKTGMAYPHTSHCLASGEIMISCLGNKDGN 160

MsSBP_Alfalfa TKTNPRAPSLHKVVEPEDISTKTGLAYPHTSHCLASGEIMISCIGDKDGN 173

Tc1SBP1_Cacao TQTNPKAPSLHKVVDPEDIVQKTGLAYPHTSHCLASGDIMVSCLGDKDGN 161

Cs1SBP2_Cucumber TKSNPTAPSLHKVVEPEDIVQKTGLAFPHTSHCLASGDIMVSCLGDKDGN 166

Pt1SBP_Poplar TLKDPRAPSLHKVVEPADIVNKTGLAYPHTSHCLASGDVMVSCLGDKDGN 168

RcSBP_Castor_bean TQKDPKAPSLHKVVEPADIIQKTGLAYPHTSHCLGSGDIMISCLGDKDGK 160

VvSBP1_Wine_grape TQKNPRGPSLHKVVEPEDILKKTGLAYPHTAHCLASGDIMVSCLGDGDGK 158

Cs2SBP2S_Weet_orange TQKNPRAPSLHKVVEPADIVQKTGLSYPHTAHCLASGDILVSCLGDKDGN 172

StSBP1_Potato TQKDPKAPSLYKVVQPDDVIKKTGLAFPHTAHCLASGEIMLSCLGDKDGN 172

BmSBP1_Yak VATEPRAPKLHKVVEPKEIHAKCDLSYLHTSHCLASGEVMISALGDPKGN 186

TtSBP1_Dolphin VGTEPRAPRLHKVVEPKDIHAKCDLGYLHTTHCLASGDVMISSLGDPKGN 158

HsSBP1_Human VGSEPRAPKLHKVIEPKDIHAKCELAFLHTSHCLASGEVMISSLGDVKGN 200

Pt2SBP1_Chimpanzee VGSEPRAPKLHKVIEPEDIHAKCELAFLHTSHCLASGEVMISSLGDVKGN 200

GggSBP1_Western_lowland_gorila VGSEPRAPKLHKVIEPKDIHAKCELAFLHTSHCLASGEVMISSLGDVKGN 158

LaSBP1_Elephant VASEPRAPKLHKIIEPKDIHAKCGLGYLHTSHCLASGEVMISSLGDPNGN 170

MmSBP1_House_mouse VGSEPRAPKLHKVIEASEIQAKCNVSSLHTSHCLASGEVMVSTLGDLQGN 158

RnSBP1_Norway_rat VGSEPRAPKLHKVIEPNEIHAKCNLGNLHTSHCLASGEVMISSLGDPQGN 158

HgSBP1_Naked_mole-rat VGSQSRAPKLHKVIEPQEVHAKCNLGNLHTSHCLPSGEVMISSLGDPKGN 157

ShSBP1_Tasmanian_devil VATEPRAPKLHKVVEPTEVMSKCDLAYLHTSHCLPSGEVMISALGDPKGN 157

. .:. .* *:*:::. :: * :. **:*** **::::* :*: .*:

GmSBP1_Soybean AAGNGFLLLDSE-FNVKGRWEKPGHSPLFGYDFWYQPRHNTMISTSWGAP 214

PvSBP_Bean AEGNGILLLDSE-FNVKRKGEKPGHSPQFGYDFWYQPRPNTMISTSWGAP 209

MsSBP_Alfalfa AEGNGFLLLDSE-FNVKGRWEKPGHSPLFGYDFWYQPRHNTMISTSWGAP 222

Tc1SBP1_Cacao AKGNGFLLLDSE-FNVKGRWEKPGHSPLFGYDFWYQPRHKTMISSSWGAP 210

Cs1SBP2_Cucumber AQGNGFLLLDSE-FNVKGRWEKPGNSPLFGYDFWYQPRHKTMISSSWGAP 215

Pt1SBP_Poplar AEGNGFLLLDSE-FNVKGRWEKPGHSPTFGYDFWYQPRHNIMISSSWGAP 217

RcSBP_Castor_bean AEGNGFLLLDSN-FNVKGRWEKPGHSPLFGYDFWYQPRHNTMISSSWGAP 209

VvSBP1_Wine_grape AEGSGFLLLDSE-FNVKGRWEKPGHSPSFGYDFWYQPRHKTMISSSWGAP 207

Cs2SBP2S_Weet_orange AEGNGFLLLDSE-FNVKGRWEKPGHSPAFGYDFWYQPRHKTMISSSWGAP 221

StSBP1_Potato AEGNGFLLLDSD-FNVKGRWEKPGHSPLFGYDFWYQPRHNTMISSTWGAP 221

BmSBP1_Yak GKG-GFVLLDGETFEVKGTWEQPGGAAPMGYDFWYQPRHNVMISTEWAAP 235

TtSBP1_Dolphin GKG-GFVLLDGETFEVKGTWERPGGAAPMGYDFWYQPRHNVMVSTEWAAP 207

HsSBP1_Human GKG-GFVLLDGETFEVKGTWERPGGAAPLGYDFWYQPRHNVMISTEWAAP 249

Pt2SBP1_Chimpanzee GKG-GFVLLDGETFEVKGTWERPGGAAPSGYDFWYQPRHNVMISTEWAAP 249

GggSBP1_Western_lowland_gorila GKG-GFVLLDGETFEVKGTWERPGGAAPLGYDFWYQPRHNVMISTEWAAP 207

LaSBP1_Elephant AKG-GFVLLDGETFEVKGTWEKPGGAAPMGYDFWYQPRHNVMISTEWAAP 219

MmSBP1_House_mouse GKG-SFVLLDGETFEVKGTWEKPGDAAPMGYDFWYQPRHNVMVSTEWAAP 207

RnSBP1_Norway_rat GKG-GFVLLDGETFEVKGTWEKPGGEAPMGYDFWYQPRHNIMVSTEWAAP 207

HgSBP1_Naked_mole-rat AKG-GFVLLDGETFQVKGTWERPRSAAPMGYDFWYQPRHNVMISTEWAAP 206

ShSBP1_Tasmanian_devil AKG-GFLLLDGETFEVKGTWQKPGGAARMGYDFWYQPRHNVLISSEWAAP 206

. * .::***.: *:** ::* . ********* : ::*: *.**

GmSBP1_Soybean SAFTKGFNLQHLSDGLYGRHLHVYSWPGGELRQTLDLGDSGLLPLEIRFL 264

PvSBP_Bean RAFTKGFNLEHLFEGLYGRHLHVYDWPGGELRQTLDLGDSGLLPLEIRFL 259

MsSBP_Alfalfa KAFLQGFNLQHVADGLYGRHLHVYSWPGGEIKQTLDLGDKGLLPLEIRFL 272

Tc1SBP1_Cacao AAFTKGFNLQHVADGLYGRHLYVYSWPDGELKQTLDLGDSGLLPLEIRFL 260

Cs1SBP2_Cucumber SAFIKGFNLQHVADGLYGKHLHVYSWPDGELKQTLDLGDTGLLPLETRFL 265

Pt1SBP_Poplar AAFTKGFNLQHVADGLYGRHLNVYSWPNGELKQTLDLGDTGLLPLEIRFL 267

RcSBP_Castor_bean AAFSKGFDLQHVSDGLYGRHLHVYSWPNGELKQTLDLGNTGLLPLEIRFL 259

VvSBP1_Wine_grape AAFTKGFNLQHVSDGLYGKHLYVYSWPEGELKQTLDLGDSGLLPLEIRFL 257

Cs2SBP2S_Weet_orange LAFTKGFNLQHVSDGLYGRHLFVYSWPDGELKQTLDLGNTGLIPLEIRFL 271

StSBP1_Potato SAFTKGFNLQDVADGHYGRHLHVYTWPGGELKQTLDLGNTGLLPLEVRFL 271

BmSBP1_Yak NVLRDGFNPADVEAGLYGQHLHVWDWQRHEKVQTLTLQD-GLIPLEIRFL 284

TtSBP1_Dolphin NVLRDGFNPADVEAGLYGNHLHVWDWQRHEMVQTLTLQD-GLIPLEVRFL 256

HsSBP1_Human NVLRDGFNPADVEAGLYGSHLYVWDWQRHEIVQTLSLKD-GLIPLEIRFL 298

Pt2SBP1_Chimpanzee NVLRDGFNPADVEAGLYGSHLYVWDWQHHEIVQTLSLKD-GLIPLEIRFL 298

GggSBP1_Western_lowland_gorila NVLRDGFNPADVEAGLYGSHLYVWDWQRHEIVQTLSLKD-GLIPLEIRFL 256

LaSBP1_Elephant NVLLDGFNPADVEAGLYGSHLHVWDWQRHEIVQTLPLQD-GLIPLEIRFL 268

MmSBP1_House_mouse NVFKDGFNPAHVEAGLYGSRIFVWDWQRHEIIQTLQMTD-GLIPLEIRFL 256

RnSBP1_Norway_rat NVFKDGFNPAHVEAGLYGSHIHVWDWQRHEIIQTLQMKD-GLIPLEIRFL 256

HgSBP1_Naked_mole-rat NVFKDGFNPAHVKDGLYGSKLHIWDWQRHELIQTLPMKD-GLIPLEVRFL 255

ShSBP1_Tasmanian_devil NVFKDGFNPADVSVGLYGSQLHVWDWQRRELIQTLQLED-GLIPLEIRFL 255

.: .**: .: * ** :: :: * * *** : : **:*** ***

62

GmSBP1_Soybean HDPAKDTGFVGSALTSNMIRFFKTQDESWSHEVAISVKPLKVQNWILPEM 314

PvSBP_Bean HDPAKDTGFVGSALTSNMIRFFKTQDGSWSHEVSISVKPLKVQNWILPEM 309

MsSBP_Alfalfa HDPAKDTGFVGSALTSNMIRFFKTQDGSWNHEIVISVEPLKVQNWFLPEM 322

Tc1SBP1_Cacao HDPSKDTGFVGCALTSNMVRFFKTKDGSWSHEVAISVKPLKVQNWILPEM 310

Cs1SBP2_Cucumber HDPSKDTGYVGCALTSNMVRFYKNQDDTWSHEVSISVKALKVQNWILPEM 315

Pt1SBP_Poplar HDPSKDSGFVGCALTSNMVRFFKTPDGSWSHEVAISVKPLKVQNWILPEM 317

RcSBP_Castor_bean HDPSKDTGFVGCALTSNMVRFFKTPDGSWSHEVAISVKPLKVKNWILPEM 309

VvSBP1_Wine_grape HDPSKDTGFVGCALTSNMVRFFKTPDGSWSHEVAISVKPLKVQNWILPDM 307

Cs2SBP2S_Weet_orange HDPSKDIGFVGCALASTMVRFFKTQDGSWNHEVAISVKSLKVQNWILPEM 321

StSBP1_Potato HNPSEAIGYVGCALTSNMVRFFKNPDGSWGHEVAISVKPVKVQNWILPEM 321

BmSBP1_Yak HNPAADQGFVGCALGSNIQRFYKNQGGTWSVEKVIQVPPKKVKGWILPEM 334

TtSBP1_Dolphin HNPAADQGFVGCALSSNIQRFYKNEGGTWSVEKVIQVPPKKVKGWMLPEM 306

HsSBP1_Human HNPDAAQGFVGCALSSTIQRFYKNEGGTWSVEKVIQVPPKKVKGWLLPEM 348

Pt2SBP1_Chimpanzee HNPDAAQGFVGCALSSTIQRFYKNEGGTWSVEKVIQVPPKKVKGWLLPEM 348

GggSBP1_Western_lowland_gorila HNPDAAQGFVGCALSSTIQRFYKNEGGTWSVEKVIQVPPKKVKGWLLPEM 306

LaSBP1_Elephant HNPDATQGFVGCALSTNIQRFYKNEGGTWSVEKVIQVAPKKVKGWMLPEM 318

MmSBP1_House_mouse HDPSATQGFVGCALSSNIQRFYKNAEGTWSVEKVIQVPSKKVKGWMLPEM 306

RnSBP1_Norway_rat HDPDATQGFVGCALSSNIQRFYKNEGGTWSVEKVIQVPSKKVKGWMLPEM 306

HgSBP1_Naked_mole-rat HDPDANEGFVGCALSSSIQRFYQNKAATWSVEKVIQVPPKKVTGWMLPEM 305

ShSBP1_Tasmanian_devil HDPAASQGFVGCALSSNIQRFYKTEGGKWAIEKVIQVPSKKVEGWMLPDM 305

*:* *:**.** :.: **::. .* * *.* . ** .*:**:*

GmSBP1_Soybean PGLITDFLISLDDRFLYFVNWLHGDIRQYNIENLKNPKLTGQVWVGGLIQ 364

PvSBP_Bean PGLITDFLISLDDRFLYFVNWLHGDIRQYNIEDIKNPKLTGQVWVGGLIQ 359

MsSBP_Alfalfa PGLITDFLISLDDRFLYFVNWLHGDIRQYNIEDVKNPKLTGQVWAGGLIQ 372

Tc1SBP1_Cacao PGLITDFLISLDDRFLYFANWLHGDVRQYNIEDPKNPVLAGQVWVGGLIQ 360

Cs1SBP2_Cucumber PGLITDFLISLDDRFLYFVNWLHGDVRQYNIEDPKSPKLVGQVWVGGLIQ 365

Pt1SBP_Poplar PGLVTDFLISLDDRFLYFVNWLHGDVRQYSIEDPEKPVLKGQVWVGGLIQ 367

RcSBP_Castor_bean PGLITDFLISLDDRFLYFVNWLHGDIRQYNIEDLKNPVLTGQVWVGGLLQ 359

VvSBP1_Wine_grape PSLITDFLISLDDRYLYLANWLHGDVRQYNIEDPKNPVLTGQVWVGGLIQ 357

Cs2SBP2S_Weet_orange PGLITDFLISLDDRFLYFSNWLHGDIRQYNIEDPKNPVLTGQVWVGGLFQ 371

StSBP1_Potato PGLITDFLISLDDRFLYLANWLHGDIRQYNIEDPANPKLTGQVFVGGVFQ 371

BmSBP1_Yak PSLITDILLSLDDRFLYFSNWLHGDLRQYDISDPKRPRLVGQIFLGGSIV 384

TtSBP1_Dolphin PGLITDILLSLDDRFLYFSNWLHGDLRQYDISDPQRPRLTGQLFLGGSIV 356

HsSBP1_Human PGLITDILLSLDDRFLYFSNWLHGDLRQYDISDPQRPRLTGQLFLGGSIV 398

Pt2SBP1_Chimpanzee PGLITDILLSLDDRFLYFSNWLHGDLRQYDISDPQRPRLTGQLFLGGSIV 398

GggSBP1_Western_lowland_gorila PGLITDILLSLDDRFLYFSNWLHGDLRQYDISDPQRPRLTGQLFLGGSIV 356

LaSBP1_Elephant PGLITDILLSLDDRFLYFSNWLHGDLRQYDISDPKKPRLAGQLFLGGSIV 368

MmSBP1_House_mouse PGLITDILLSLDDRFLYFSNWLHGDIRQYDISNPQKPRLAGQIFLGGSIV 356

RnSBP1_Norway_rat PGLITDILLSLDDRFLYFSNWLHGDIRQYDISNPKKPRLTGQIFLGGSIV 356

HgSBP1_Naked_mole-rat PGLITDILLSLDDRFLYFSNWLHGDVRQYDVSDPQRPRLTGQIFLGGSIV 355

ShSBP1_Tasmanian_devil PGLITDILLSLDDRFLYFSNWVHGDLRQYDISDPQRPRLVGQIFIGGSIV 355

*.*:**:*:*****:**: **:***:***.:.: * * **:: ** :

GmSBP1_Soybean KGSPVVAITDDGETWQAEVPEIQGNKLRGGPQMIQLSLDGKRLYATNSLF 414

PvSBP_Bean KGSPIIAVTDDGETWQAEVPEIQGKKLRAGPQMIQLSLDGKRLYATNSLF 409

MsSBP_Alfalfa KGSPVVAVKDDGETWQSDVPEIQGKKLRGGPQMIQLSLDGKRLYVTNSLF 422

Tc1SBP1_Cacao NGSPVVAVIEDGKTWQCNVPEIQGHRLRGGPQMIQLSLDGKRLYVTNSLF 410

Cs1SBP2_Cucumber KGSPVLAEAEDGTTFQFDVPEIKGQRLRGGPQMIQLSLDGKRLYVTNSLF 415

Pt1SBP_Poplar KGSSVVAEGEDGKTWQYDVPEIQGHRLRGGPQMIQLSLDGKRLYVTNSLF 417

RcSBP_Castor_bean KGSPIMVETEDRSTWQADVPEIQGNRLRGGPQMIQLSLDGKRLYVTNSLF 409

VvSBP1_Wine_grape KGSPIVALAEDGTTWQSEVPEVQGKRLRGGPQMIQLSLDGKRLYVTNSLF 407

Cs2SBP2S_Weet_orange KGSPVVAVTDDGQPYQSDVPSVQGHRLRGGPQMIQLSLDGKRLYVTNSLF 421

StSBP1_Potato KGNAVLAEAEDGSTYQVDVPEVQGHRLRGGPQMIQLSLDGKRLYATNSLF 421

BmSBP1_Yak KGGPVQVLEDQELKCQPEPLVVKGKRVAGGPQMIQLSLDGTRLYVTTSLY 434

TtSBP1_Dolphin KGGPVQVLEDQELKSQPEPLVVKGKQVAGGPQMIQLSLDGKRLYVTTSLY 406

HsSBP1_Human KGGPVQVLEDEELKSQPEPLVVKGKRVAGGPQMIQLSLDGKRLYITTSLY 448

Pt2SBP1_Chimpanzee KGGPVQVLEDQELKSQPEPLVVKGKRVAGGPQMIQLSLDGKRLYITTSLY 448

GggSBP1_Western_lowland_gorila KGGPVQVLEDQELKSQPEPLVVKGKRVAGGPQMIQLSLDGKRLYITTSLY 406

LaSBP1_Elephant KGGPVQVLEDQELESQPEPLVVKGKHVAGGPQMIQLSLDGKRLYVTTSLY 418

MmSBP1_House_mouse RGGSVQVLEDQELTCQPEPLVVKGKRIPGGPQMIQLSLDGKRLYATTSLY 406

RnSBP1_Norway_rat KGGSVQVLEDQELTCQPEPLVVKGKRVPGGPQMIQLSLDGKRLYVTTSLY 406

HgSBP1_Naked_mole-rat KGGPVQVLEDQELKCQPDPLVVKGKRVAGGPQMIQLSLDGKRLYVTTSLY 405

ShSBP1_Tasmanian_devil QGGPVRVLEDKELKCQPVPLVVKGKKIQGGPQMIQLSLDGRRLYVTTSLY 405

.*..: . :. * ::*::: .*********** *** *.**:

GmSBP1_Soybean STWDKQFYPELVQKGSHIIQIDVDTEKGGLKINPNFFVDFGAEPDGPSLA 464

PvSBP_Bean STWDKQFYPDLVQQGSHIIQIDVDTQKGGLKINPNFFVDFGTEPHGPSLA 459

MsSBP_Alfalfa SAWDKQFYPKLVEQGSHILQIDVDTENGGLKINPNFFVDFGAEPDGPSLA 472

Tc1SBP1_Cacao STWDRQFYPELVEKGSHMLQIDVDTEKGGLKVNPYFFVDFGAEPDGPSLA 460

Cs1SBP2_Cucumber STWDRQFYPELVEKGSHMLQIDVDTQKGGLSVNPNFFVDFATEPDGPSLA 465

Pt1SBP_Poplar STWDRQFYPELMEKGSHMLQIDVDTEKGGLAINPNYFVDFAAEPDGPSLA 467

RcSBP_Castor_bean STWDRQFYPELVEKGSHMLQIDVDTEKGGLKVNPNFFVDFAAEPDGPSLA 459

VvSBP1_Wine_grape STWDHQFYPDLPREGSHMLQIDVDTEKGGLVINPNFFVDFGSEPDGPSLA 457

Cs2SBP2S_Weet_orange SAWDCQFYPELKEKGSHMLQIDVNSEKGGMAINPNFFVDFEAEPDGPALA 471

StSBP1_Potato STWDRQFYPEMVEKGGHMLQIDVDSEKGGLAINPRFFVDFGAEPDGPSLA 471

BmSBP1_Yak SAWDKQFYPDLIREGSVMLQIDVDTVRGGLKLNPNFLVDFGKEPLGPALA 484

63

TtSBP1_Dolphin SAWDKQFYPDLIREGSVMLQIDVDTVKGGLKLNPNFLVDFGKEPLGPALA 456

HsSBP1_Human SAWDKQFYPDLIREGSVMLQVDVDTVKGGLKLNPNFLVDFGKEPLGPALA 498

Pt2SBP1_Chimpanzee SAWDKQFYPDLIREGSVMLQVDVDTVKGGLKLNPNFLVDFGKEPLGPALA 498

GggSBP1_Western_lowland_gorila SAWDKQFYPDLIREGSVMLQVDVDTVKGGLKLNPNFLVDFGKEPLGPALA 456

LaSBP1_Elephant SAWDKQFYPDLIREGSVMLQIDVNTEQGGLKLNPNFLVDFGKEPHGPALA 468

MmSBP1_House_mouse SAWDKQFYPDLIREGSMMLQIDVDTVNGGLKLNPNFLVDFGKEPLGPALA 456

RnSBP1_Norway_rat SAWDKQFYPNLIREGSVMLQIDVDTANGGLKLNPNFLVDFGKEPLGPALA 456

HgSBP1_Naked_mole-rat SAWDKQFYPDLIREGSVMLQVDVDTERGGLKLNPNFLVDFGKEPLGPALA 455

ShSBP1_Tasmanian_devil SAWDKQFYPDLIKEGSVMLQVDVDTEKGGLTLNPDFLVDFGKEPLGPALA 455

*:** ****.: .:*. ::*:**:: .**: :** ::*** ** **:**

GmSBP1_Soybean HEMRYPGGDCTSDIWI 480

PvSBP_Bean HEMRYPGGDCTSDIWI 475

MsSBP_Alfalfa HEMRYPGGDCTSDIWI 488

Tc1SBP1_Cacao HEMRYPGGDCTSDIWI 476

Cs1SBP2_Cucumber HEMRYPGGDCTSDIWI 481

Pt1SBP_Poplar HEMRYPGGDCTSDIWI 483

RcSBP_Castor_bean HEMRYPGGDCTSDIWI 475

VvSBP1_Wine_grape HEMRYPGGDCTSDIWV 473

Cs2SBP2S_Weet_orange HEMRYPGGDCTSDIWI 487

StSBP1_Potato HEMRYPGGDCTSDIWI 487

BmSBP1_Yak HELRYPGGDCSSDIWL 500

TtSBP1_Dolphin HELRYPGGDCSSDIWL 472

HsSBP1_Human HELRYPGGDCSSDIWI 514

Pt2SBP1_Chimpanzee HELRYPGGDCSSDIWI 514

GggSBP1_Western_lowland_gorila HELRYPGGDCSSDIWI 472

LaSBP1_Elephant HELRYPGGDCSSDIWI 484

MmSBP1_House_mouse HELRYPGGDCSSDIWI 472

RnSBP1_Norway_rat HELRYPGGDCSSDIWI 472

HgSBP1_Naked_mole-rat HEMRYPGGDCTSDIWI 471

ShSBP1_Tasmanian_devil HELRYPGGDCSSDIWL 471

**:*******:****:

Supplementary material 4. Alignment of amino acid sequences of TcGPXs with others plants and

mammalians GPXs. The sequences used for alignment are: TcPHGPx, Theobroma cacao

(XP_007011699.1); GaPHGPx, Gossypium arboreum (KHG09053.1); AtPHGPx, Arabidopsis

thaliana (NP_180080.1); VvGPx2, Vitis vinifera (XP_002263327.1); TcGPx2, Theobroma cacao

(XP_007026518.1); PeGPx2, Populus euphratica (XP_011026385.1); GmGPx4, Glycine

max (NP_001238132.1); TcGPx4, Theobroma cacao (XP_007050669.1); PtGPx4, Populus

trichocarpa (XP_002320392.1); GmGPx6, Glycine max (KHN19874.1); TcGPx6, Theobroma

cacao (XP_007040204.1); FvGPx6, Fragaria vesca (XP_004298758.1); TcGPx8, Theobroma

cacao (XP_007040205.1); CsGPx8, Citrus sinensis (XP_006476628.1); SlGPx8, Solanum

lycopersicum (XP_004252596.1); MmGPX, Mus musculus (NP_032186.2); BtGPX, Bos

taurus (1GP1_A); EcGPX, Equus caballus (NP_001159951.1); HsGPX, Homo

sapiens (NP_000572.2); MmGPX, Macaca mulata (NP_001152770.1). Gaps introduced to get the

best alignment are indicated by (-), (*) represents identical amino acids between all sequences, (.) and

(:) represent conserved substitutions and semi-conserved substitutions, respectively.

TcGPX8_Cacao --------------------------------------------------

CsGPX8_Orange --------------------------------------------------

SlGPX8_Tomato --------------------------------------------------

TcGPX6_Cacao MLCSPT--YLFRRNLSAVAVSASLLLSKR---LSPSSKQTLLSFPQISPV 45

FvGPX6_Freesia MLCSSSR-LVFRRNL---AVAAALVFPRQ---FSTVSKNTLLRPSCFP-- 41

CaGPX6_Grain_peas MLCTSTRFFLLTTSTARLALAAPLSSSSSSYFFSTNNYYNPISFSSLPNK 50

TcPHGPX_Cacao -MASMPFSATFPSYLHDLSQTKKIPVMPSSWP---FSIPSIESSLGSSKS 46

GaPHGPX_Coton -MASMSFSATIPSPLLDFSQTKKNQVFSSSWPSMSFSIPSIKSSLGSSKS 49

AtPHGPX_Thale_cress -MVSMTTSS---SSYGTFSTVVNSSRPNSSAT---FLVPSLKFSTGISNF 43

TcGPX2_Cacao ------------------------------------MMHWLRFTNLVS-- 12

VvGPX2_Wine_grape --------------------------------------------------

PeGPX2_Euphrates_poplar ----------------------------------MLAFYKMHFTNTIS-- 14

TcGPX4_Cacao --------------------------------------------------

PtGPX4_Poplar --------------------------------------------------

GmGPX4_Soybean --------------------------------------------------

HsGPX_Human --------------------------------------------------

MmGPX_Rhesus_monkey --------------------------------------------------

EcGPX_Horse --------------------------------------------------

64

BtGPX_cattle --------------------------------------------------

MmGPX_House_mouse --------------------------------------------------

TcGPX8_Cacao ------------------------MASQSTKNPESIYDFTVKDAKGNV-V 25

CsGPX8_Orange ------------------------MTSQFIQNPESIFDLSVKDARGHE-V 25

SlGPX8_Tomato ------------------------MAGQPEKKPQSVYDFSLKDATGND-V 25

TcGPX6_Cacao SLVSPSIETGFSRSFLGSLRFDHIMAGQSSK--GSIHDFTVKDARGND-V 92

FvGPX6_Freesia SIKAQSFNP------VSSFQFNRSMASQSSQP-KTVHDFTVKDARGND-V 83

CaGPX6_Grain_peas PKPNSKPLLTTSTSLYFTLRADHTMASQSNP--NSIHDFTVKDAKGND-V 97

TcPHGPX_Cacao GFLQHGFSLQSSSVPGFVFKSRSSGIYARAATEKTLYDYTVKDIDGKD-V 95

GaPHGPX_Coton AFFQNGFSLLSLTASGFVFNSRSSGIYARAATDKTLYDYTVKDIDGKD-T 98

AtPHGPX_Thale_cress ANLSNGFSLKSPINPGFLFKSRPFTVQARAAAEKTVHDFTVKDIDGKD-V 92

TcGPX2_Cacao -LVFLGFAFFLY----FHIYPSSSHQNMAENAPKSVYEFTVKDIRGND-V 56

VvGPX2_Wine_grape ---------------------------MAEAAPKSIYDFTVKDIRGND-V 22

PeGPX2_Euphrates_poplar -LVFLGFAILAL----YSYPSLIPSRKMAEESPKSIYDFTVKDIRGND-T 58

TcGPX4_Cacao ------------------------MGASESVPQKSIHQFTVKDNKGQD-V 25

PtGPX4_Poplar ------------------------MGSSPSVPEKSIHEFTVKDNRGQD-V 25

GmGPX4_Soybean ------------------------MGASQSISENSIHEFTVKDARGKD-V 25

HsGPX_Human ---------------MCAAR-----LAAAAAAAQSVYAFSARPLAGGEPV 30

MmGPX_Rhesus_monkey ---------------MCAAR-----LAAAA-----VYAFSARPLAGGEPV 25

EcGPX_Horse ---------------MCAAQ-----LAAAAR--RSVYAFSARPLAGGEPL 28

BtGPX_cattle ---------------MCAAQRSAAALAAAAP--RTVYAFSARPLAGGEPF 33

MmGPX_House_mouse ---------------MCAAR-----LSAAAQ--STVYAFSARPLTGGEPV 28

:. : : *

TcGPX8_Cacao DLSAYKGKVLLIVNVASKWYLRSDSVINTGRLFFATDGMTNPNYTELNQL 75

CsGPX8_Orange DLSTYKGKVLLIVNVASKC------------------GMTNSNYIELSQL 57

SlGPX8_Tomato DLSIFKGKVLLIVNVASKC------------------GMTNSNYTELNQL 57

TcGPX6_Cacao DLSIYKGKVLLIVNVASQC------------------GLTNSNYTELSQL 124

FvGPX6_Freesia DLSTYKGKVLLIVNVASQC------------------GLTNSNYTELAQL 115

CaGPX6_Grain_peas NLGDYKGKVLLIVNVASQC------------------GLTNSNYTELSQL 129

TcPHGPX_Cacao SLSRFKGKVLLIVNVASKC------------------GLTTSNYSELSHI 127

GaPHGPX_Coton PLSKFKGKVLLIVNVASRC------------------GLTTSNYSELSHI 130

AtPHGPX_Thale_cress ALNKFKGKVMLIVNVASRC------------------GLTSSNYSELSHL 124

TcGPX2_Cacao SLSEYSGKVLLIVNVASKC------------------GLTHSNYKELNVL 88

VvGPX2_Wine_grape SLSDYNGKVLLIVNVASKC------------------GLTHSNYKELNVL 54

PeGPX2_Euphrates_poplar SLSEYSGKVLLIVNVASKC------------------GLTHSNYKELNVL 90

TcGPX4_Cacao DLSIYEGKVLLVVNVASKC------------------GLTDSNYTQLTDL 57

PtGPX4_Poplar NLGIYKGKVLLVVNVASKC------------------GFTDSNYTQLTDL 57

GmGPX4_Soybean NLNAYRGKVLLVINVASKC------------------GFADANYSQLTQI 57

HsGPX_Human SLGSLRGKVLLIENVASLG-------------------TTVRDYTQMNEL 61

MmGPX_Rhesus_monkey SLGSLRGKVLLIENVASLG-------------------TTVRDYTQMNEL 56

EcGPX_Horse SLGSLRGKVLLIENVASLG-------------------TTVRDYTQMNEL 59

BtGPX_cattle NLSSLRGKVLLIENVASLG-------------------TTVRDYTQMNDL 64

MmGPX_House_mouse SLGSLRGKVLLIENVASLG-------------------TTIRDYTEMNDL 59

*. ***:*: **** : :* :: :

TcGPX8_Cacao YEKYKDQGLEILAFPCNQFGEEEPGSNDQIAVFVCTR-----FRSEFPIF 120

CsGPX8_Orange YDKYKDQGLEILAFPCNQFGEEEPGSNDQIADFVCTR-----FKSEFPIF 102

SlGPX8_Tomato YEKYKDQGLEILAFPCNQFGEEEPGTNDQILNFVCTR-----FKSDFPIF 102

TcGPX6_Cacao YEKYKDQGLEILAFPCNQFGGQEPGNNEQILEFACTR-----FKAEYPIF 169

FvGPX6_Freesia YEKYKTQGLEILAFPCNQFGAQEPGSNDEIVEFACTR-----FKAEYPIF 160

CaGPX6_Grain_peas YDKYKQKGLEILAFPCNQFGAQEPGSLEEIQDFVCTR-----FKAEFPVF 174

TcPHGPX_Cacao YEKYKTQGFEILAFPCNQFGGQEPGSNPEIKQFACTR-----FKAEFPIF 172

GaPHGPX_Coton YDKYKNQGFEILAFPCNQFGGQEPGSNPDIKKFACTR-----FKAEFPIF 175

AtPHGPX_Thale_cress YEKYKTQGFEILAFPCNQFGFQEPGSNSEIKQFACTR-----FKAEFPIF 169

TcGPX2_Cacao YEKYKNQGFEILAFPCNQFAGQEPGTNEHIQEVACTM-----FKAEFPIF 133

VvGPX2_Wine_grape YEKYKSQGFEILAFPCNQFLGQEPGSNEEILEAACTM-----FKAEFPIF 99

PeGPX2_Euphrates_poplar YEKYKNQGFEILAFPCNQFAGQEPGSNEEIQDTVCTI-----FKAEFPIF 135

TcGPX4_Cacao YSKYKDQGLEILAFPCNQFLKQEPGTEQEVQQFACTR-----YKAEYPIF 102

PtGPX4_Poplar YKNYKDKGLEILAFPCNQFLNQEPGTSEDAQNFACTR-----YKADYPIF 102

GmGPX4_Soybean YSTYKSRGLEILAFPCNQFLKKEPGTSQEAQEFACTR-----YKAEYPIF 102

HsGPX_Human QRRLGPRGLVVLGFPCNQFGHQENAKNEEILNSLKYVRPGGGFEPNFMLF 111

MmGPX_Rhesus_monkey QRRLGPRGLVVLGFPCNQFGHQENAKNEEILNSLKYVRPGGGFEPNFMLF 106

EcGPX_Horse QRRLGPRGLVVLGFPCNQFGHQENAKNEEILNSLKYVRPGGGFEPNFTLF 109

BtGPX_cattle QRRLGPRGLVVLGFPCNQFGHQENAKNEEILNCLKYVRPGGGFEPNFMLF 114

MmGPX_House_mouse QKRLGPRGLVVLGFPCNQFGHQENGKNEEILNSLKYVRPGGGFEPNFTLF 109

:*: :*.****** :* .. . :..:: :*

TcGPX8_Cacao DKIEVNGDNASPLYKYLKLGK-------------------WGIFGDDIQW 151

CsGPX8_Orange EKIDVNGEHASPLYKLLKSGK-------------------WGIFGDDIQW 133

SlGPX8_Tomato DKIEVNGENASPLYKFLKSGK-------------------WGIFGDDIQW 133

TcGPX6_Cacao DKVDVNGEKTAPIYKFLKSSK-------------------GGLFGDSIKW 200

FvGPX6_Freesia DKVDVNGDKATPLYKFLKSSK-------------------GGLFGDSIKW 191

CaGPX6_Grain_peas DKVDVNGDSAAPIYKYLKSSK-------------------GGLFGDNIKW 205

TcPHGPX_Cacao DKVDVNGPNTAPVYQFLKSNA-------------------GGFLGDLVKW 203

65

GaPHGPX_Coton DKVDVNGPNTAPVYQFLKSSA-------------------GGFFSDLIKW 206

AtPHGPX_Thale_cress DKVDVNGPSTAPIYEFLKSNA-------------------GGFLGGLIKW 200

TcGPX2_Cacao DKVEVNGKNSAPLYKFLKSVK-------------------GGYFGDAIKW 164

VvGPX2_Wine_grape DKVEVNGKNTAPLYKFLKLQK-------------------GGLFGDGIKW 130

PeGPX2_Euphrates_poplar DKIDVNGKNTAPVYKFLKSEK-------------------GGYFGDAIKW 166

TcGPX4_Cacao RKVRVNGPKTEPVYKFLKSNK-------------------SGFLGSRIKW 133

PtGPX4_Poplar HKVRVNGPNAAPVYKFLKASK-------------------PGFLGNRIKW 133

GmGPX4_Soybean GKIRVNGSDTAPVFKFLKTQK-------------------SGVMGSRIKW 133

HsGPX_Human EKCEVNGAGAHPLFAFLREALPAPSDDATALMTDPKLITWSPVCRNDVAW 161

MmGPX_Rhesus_monkey EKCEVNGAGAHPLFAFLREALPAPSDDATALMTDPKLITWSPVCRNDVAW 156

EcGPX_Horse EKCEVNGAQAHPLFAFLREALPAPSDDATALMTDPKFITWSPVCRNDVAW 159

BtGPX_cattle EKCEVNGEKAHPLFAFLREVLPTPSDDATALMTDPKFITWSPVCRNDVSW 164

MmGPX_House_mouse EKCEVNGEKAHPLFTFLRNALPTPSDDPTALMTDPKYIIWSPVCRNDIAW 159

* *** : *:: *: . : *

TcGPX8_Cacao NFAKFLVSKDGQVVHRYYPTTSPLSLEY-------DIKKLLGLGQE---- 190

CsGPX8_Orange NFAKFLVDKNGQVVDRYYPTTSLLSLEH-------DIKKLLGLS------ 170

SlGPX8_Tomato NFAKFLVDKNGQVVDRYYPTTSPLTIER-------DMKKLLETI------ 170

TcGPX6_Cacao NFSKFLVDKEGNVVDRYAPTTSPLSIEK-------DIKKLLA-------- 235

FvGPX6_Freesia NFSKFLVDKEGNVVNRYAPTTTPLSIEK-------DVKKLLGVA------ 228

CaGPX6_Grain_peas NFSKFLVDKNGNVVERYAPTTSPLSIEK-------DLLKLLGA------- 241

TcPHGPX_Cacao NFEKFLVDKNGKVVERYPPTISPFQIEK-------DIQKLLAA------- 239

GaPHGPX_Coton NFEKFLVDKNGKVVERYPPTTSPFQIEK-------DIQKLLAT------- 242

AtPHGPX_Thale_cress NFEKFLIDKKGKVVERYPPTTSPFQIEK-------DIQKLLAA------- 236

TcGPX2_Cacao NFTKFLVDKEGKVVERYAPTTSPLKIEQRTRDIAWNFQGVHGSKAWLVFG 214

VvGPX2_Wine_grape NFTKFLVDKEGKVVDRYAPTTSPLKIEE-------DIQNLLGSA------ 167

PeGPX2_Euphrates_poplar NFTKFLVNKEGKVVERYAPTTSPLKIEK-------DIQNLL--------- 200

TcGPX4_Cacao NFTKFLVDKNGHVLGRYGPTTAPLAIEA-------DIKKALGVDT----- 171

PtGPX4_Poplar NFTKFLVDKDGHVLGRYSTITAPMAIEA-------DIKKALGEM------ 170

GmGPX4_Soybean NFTKFLVDEEGRVIQRYSPTTKPLAIES-------DIKKALQVA------ 170

HsGPX_Human NFEKFLVGPDGVPLRRYSRRFQTIDIEP-------DIEALLSQGPSCA-- 202

MmGPX_Rhesus_monkey NFEKFLVGPDGVPVRRYSRRFQTIDIEP-------DIEALLSQGPSSA-- 197

EcGPX_Horse NFEKFLVGPDGVPVRRYSRRFPTIDIEP-------DIEALLTQGPSCA-- 200

BtGPX_cattle NFEKFLVGPDGVPVRRYSRRFLTIDIEP-------DIETLLSQGASA--- 204

MmGPX_House_mouse NFEKFLVGPDGVPVRRYSRRFRTIDIEP-------DIETLLSQQSGNS-- 200

** ***:. .* : ** : :* :.

TcGPX8_Cacao ------------------------------------------------

CsGPX8_Orange ------------------------------------------------

SlGPX8_Tomato ------------------------------------------------

TcGPX6_Cacao ------------------------------------------------

FvGPX6_Freesia ------------------------------------------------

CaGPX6_Grain_peas ------------------------------------------------

TcPHGPX_Cacao ------------------------------------------------

GaPHGPX_Coton ------------------------------------------------

AtPHGPX_Thale_cress ------------------------------------------------

TcGPX2_Cacao GLRLVVFISLLSNVSGWMITSSTMNYLCGAFTHRVGCKASHKIICNNL 262

VvGPX2_Wine_grape ------------------------------------------------

PeGPX2_Euphrates_poplar ------------------------------------------------

TcGPX4_Cacao ------------------------------------------------

PtGPX4_Poplar ------------------------------------------------

GmGPX4_Soybean ------------------------------------------------

HsGPX_Human ------------------------------------------------

MmGPX_Rhesus_monkey ------------------------------------------------

EcGPX_Horse ------------------------------------------------

BtGPX_cattle ------------------------------------------------

MmGPX_House_mouse ------------------------------------------------

66

Supplementary material 5. Validation of the built model of TcSBP, TcPHGPx and TcGPx2

respectively. Red, yellow, light yellow and white regions represent energetically most favored,

allowed, generously allowed and disallowed regions. The plot was generated with Anolea-swiss

model.

67

Supplementary material 6. Dissociation curves of Rt-qPCR.

68

Supplementary material 7. Characteristics of the primers designed for the qPCR study.

Gene

Primer Amplified product

Sequence Tm

(°C)

Size

(bp)

Tm

(°C) % GC GC/AT

TcSBP F: 5’-TTGTTGACTTTGGAGCTGAACCT-3’

R: 5’- TGCAGTCACCACCTGGATATCT-3’

F: 66.2

R: 66.0 71 94.6 54.9 1.21

TcPHGPX F: 5’-CTTTCTCGATTCCTTCCATTGAAT-3’

R: 5’-CCATGTTGCAAAAAGCCTGAT-3’

F: 65.4

R: 66.2 56 86.8 42.9 0.75

TcGPX2 F: 5’- TGCCTTCTTTCTGTATTTTCACATCT-3’

R: 5’-GCGTTTTCTGCCATGTTTTGA-3’

F: 65.0

R: 67.3 63 86.8 41.3 0.7

TcGPX4 F: 5’- GGTCAGGATGTGGACCTTAGCA-3’

R: 5’-CACATTTAGAAGCAACATTAACCACAA-3’

F: 67.6

R: 65.7 70 85.1 41.4 0.7

TcGPX6 F: 5’- CTTGGGTTCTTTGAGATTTGATCA-3’

R: 5’-TCATGGATTGACCCCTTGGA-3’

F: 65.4

R: 67.9 63 88 42.9 1.03

TcGPX8 F: 5’- CAATTTGGTGAGGAGGAACCA-3’

R: 5’-AGCGGGTGCAAACAAACAC-3’

F: 66.3

R: 66.4 61 90.2 45.9 0.84

69

7. CONCLUSÕES GERAIS

A proteínas TcSBP e as TcGPXs são proteínas conservadas com outras SBPs e GPXs

de espécies de mamíferos e plantas;

O motivo CSSC conservado na classe de SBPs de várias espécies é conservado

também em TcSBP;

O selenito se liga ao motivo CSSC de TcSBP;

TcSBP interage com TcPHGPx e TcGPX2;

Os genes TcSBP, TcPHGPx e TcGPx2 são diferencialmente expressos em plantas de

cacau durante a interação com M. perniciosa;

TcSBP é mais expresso na variedade Catongo tanto na fase inicial (24 a 48 horas após

inoculação) e finais (30 a 90 dias apósa inoculação);

TcGPX2 é mais expresso na variedade TSH1188 principalmente na fase inicial (24

horas após inoculação) e durante os sintomas de vassoura seca (90 dias após

inoculação);

TcPHGPx é mais expresso em Catongo com 15 dias após a inoculação;

De 30 a 90 dias, a expressão de TcPHGPx é reduzida gradativamente;

Quando TcSBP está mais expressa, a expressão de TcGPx2 é reduzida;

Quando TcGPx2 está mais expressa, a expressão de TcSBP é reduzida.