Structural and functional characterization of the Citrus sinensis protein CsMAF1, an interacting partner of the main type TAL effector of Xanthomonas citri

Orientador: Celso Eduardo BenedettiTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O cancro cítrico, causado pela bactéria Xanthomonas citri (X. citri), afeta a maioria das espécies de Citrus, ocorre praticamente em todos continentes e se destaca como uma séria ameaça à citricultura brasileira. O mecanismo molecular pelo qual X. citri causa cancro não é inteiramente conhecido, entretanto, sabe-se que a bactéria utiliza o sistema secretório tipo III para injetar proteínas de patogenicidade, entre elas, PthAs da família AvrBs3/PthA, também conhecidas como efetores TAL (transcriptional activator-like). Os efetores TAL atuam como fatores de transcrição transativando genes específicos da planta que vão beneficiar a bactéria ou desencadear respostas de defesa. Com o objetivo de entender os mecanismos moleculares pelos quais os efetores TAL atuam, a técnica de duplo híbrido foi usada para identificar proteínas de laranja doce (Citrus sinensis) que interagem com PthA4, um dos efetores TAL de X. citri necessário para o desenvolvimento do cancro cítrico. A maioria das proteínas de laranja identificadas como alvos de PthA4 apresenta domínios de ligação à DNA ou RNA e está envolvida no controle da transcrição, estabilização de mRNAs e tradução. Várias dessas proteínas interagem entre si, sugerindo a presença de um complexo multiproteico como alvo de efetores TAL. Entre as proteínas envolvidas no controle da transcrição, destacamos a CsMAF1, uma proteína homóloga à MAF1 humana que atua como regulador negativo da RNA Polimerase III. Os resultados obtidos nesse trabalho revelam que CsMAF1 complementa o fenótipo do mutante maf1 de levedura, reprimindo a expressão de tRNAHis e que a expressão de PthA4 na cepa complementada restaura a síntese desse tRNA. Portanto, os dados mostram que CsMAF1 atua como um repressor da RNA Pol III em levedura e que PthA4 altera o estado repressor de CsMAF1 sobre a RNA Pol III. De forma surpreendente, verificamos que plantas de citros com níveis reduzidos de CsMAF1 apresentaram aumento significativo no número e intensidade de lesões hiperplásticas ou eruptivas quando infiltradas com X. citri, indicando que CsMAF1 desempenha um papel crítico no desenvolvimento dos sintomas do cancro cítrico. O aumento das lesões do cancro nas plantas silenciadas para CsMAF1 se correlaciona com um aumento expressivo de tRNAs, incluindo o tRNAHis, confirmando assim o papel repressor de CsMAF1 sobre a RNA Pol III em citros. Além disso, mostramos nesse trabalho que CsMAF1 é uma fosfoproteína que se encontra na forma dimérica em solução, uma característica singular ainda não descrita para membros dessa família de proteínas. Verificamos que CsMAF1 é fosforilada in vitro pelas quinases PKA e PKC e que apresenta sítios adicionais de fosforilação conservados para a quinase TOR, incluindo o resíduo Thr62. Curiosamente, tais sítios se localizam na interface de dimerização de CsMAF1, sugerindo que a fosforilação desses sítios deve regular a função da proteína e/ou seu estado multimérico. De fato, verificamos que a substituição do resíduo de treonina Thr 62 para ácido aspártico (Asp 62) diminui a proporção dímero:monômero de CsMAF1, indicando que a fosforilação de resíduos na interface do dímero desestabiliza o dímero, e que esse pode ser um mecanismo regulatório novo para essa classe de proteína. Desse modo, esses achados abrem novas perspectivas para o entendimento não só dos mecanismos moleculares envolvidos na regulação da RNA Pol III pela CsMAF1, como também do papel de PthA4 na interação com CsMAF1 e sua modulação da transcriçãoAbstract: Citrus canker, caused by Xanthomonas citri (X. citri), is a disease that affects most of the Citrus species, occurs in almost all continents and stands as a threat to the Brazilian citrus industry. The molecular mechanism by which X. citri causes canker is poorly understood, however the bacterium injects pathogenicity proteins via the type III secretion system (T3S) including proteins of AvrBs3/PthA family, also known as transcriptional activator-like (TAL) effectors. TAL effectors have been extensively studied and are known to act as transcription factors that transactivate specific plant genes which either benefit the bacteria or trigger defense responses. To gain insights into the molecular mode of action of TAL effectors, a twohybrid screening was performed to identify sweet orange (Citrus sinensis) proteins that interact with PthA4, one of the X. citri TAL effectors required for citrus canker development. Among the proteins identified as PthA4 interactors, most are DNA and/or RNA-binding factors involved in chromatin remodeling and repair, transcriptional control and mRNA stabilization/modification. Several of these proteins interact with each other, suggesting the presence of a multiprotein complex as a target of TAL effectors. Among the proteins involved in transcription control, we selected for further studies the CsMAF1, a homolog of the human MAF1 that acts as a negative regulator of RNA polymerase III. The results presented here reveal that CsMAF1 complements the yeast maf1 mutant phenotype by repressing the tRNAHis transcription, and that PthA4 expression in the complemented strain restores the tRNAHis synthesis. Thus, the data show that CsMAF1 acts as a RNA Pol III repressor in yeast and that PthA4 somehow suppresses the repressor activity of CsMAF1 upon on the RNA Pol III. Surprisingly, we found that citrus plants with reduced levels of CsMAF1 showed a significant increase in the number, morphology and size of eruptive or hyperplastic lesions when infiltrated with X. citri, indicating the CsMAF1 plays a critical role in canker development. Increased canker lesions in CsMAF1 silenced plants correlated with a significant increase of tRNAs expression, including tRNAHis, thus confirming the repressor role of CsMAF1 upon the citrus RNA Pol III. Furthermore, we showed in this work that CsMAF1 is a phosphorylated and a dimer in solution, a feature that so far has not been reported for any member of this protein family. We found that CsMAF1 is phosphorylated in vitro by PKA and PKC, and has additional phosphorylation sites for the TOR kinase, including the Thr 62 residue. Interestingly, these phosphorylation sites are located at the dimerization interface of CsMAF1, suggesting that phosphorylation of such sites might regulate the function of the protein and / or its multimeric state. Indeed, mutation of threonine residue Thr62 to aspartic acid (Asp62) decreases the dimer:monomer CsMAF1 ratio, indicating that phosphorylation of the residues at the interface of the dimer destabilizes the dimer, and this may be a novel regulatory mechanism for this class of protein. Thus, these findings open new perspectives for the understanding of the molecular mechanisms involved in RNA Pol III regulation by CsMAF1, as well as for the role of PthA4 in the modulation of RNA Pol III transcription mediated by CsMAF1DoutoradoGenetica Vegetal e MelhoramentoDoutor em Genetica e Biologia Molecula

Crystal structure and regulation of the citrus pol III repressor MAF1 by auxin and phosphorylation

MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrusThis work was supported by Sa˜ o Paulo Research Foundation (FAPESP grant 2011/20468-1). C.E.B. and A.F.Z.N. received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Peer reviewe

The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA

Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL) effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC), a translin-associated factor X (CsTRAX), a VirE2-interacting protein (CsVIP2), a high mobility group (CsHMG) and two poly(A)-binding proteins (CsPABP1 and 2), interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U) RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control

Correction: The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA

<p>Correction: The TAL Effector PthA4 Interacts with Nuclear Factors Involved in RNA-Dependent Processes Including a HMG Protein That Selectively Binds Poly(U) RNA</p

Protein-protein interactions among the PthA4 interactors detected by yeast two-hybrid and mass spectrometry.

<p>Yeast cells double-transformed with the indicated prey-bait constructs were grown in SC -Trp -Leu -His -Ade in the presence of 5 mM 3AT. (A) Positive interactions observed between CsTRAX and CsSMC, CsPABP1, CsTRAX, CsRRMP1 and CsVIP2. (B) Protein-protein interactions observed between CsSMC and CsPABP2, CsSMC, CsPABP1, CsVIP2 and CsTRAX, but not between CsSMC and CsKH. (C) Interactions of CsVIP2 with CsKH, reciprocal interactions between CsPABP2 and CsVIP2, and self interactions of CsVIP2 and CsPABP2. (D) Weak interactions between CsHMG and the poly(A)-binding proteins CsPABP2 and CsPABP1. (E) A diagram illustrating the network of interactions observed among the citrus PthA targets. (F) Silver-stained SDS polyacrylamide gels of citrus proteins trapped in cobalt beads carrying the recombinant 6xHis-tagged CsSMC or CsTRAX as baits (bands 9 and 10, respectively). Protein bands excised from the gels, indicated by the numbers, were identified by mass spectrometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032305#pone-0032305-t002" target="_blank">Table 2</a> for details). The molecular markers (MM) are shown on the left.</p

CsHMG shows identity to plant HMGBs of group B.

<p>(A) Schematic representation of the CsHMG primary structure showing its central HMG-box domain flanked by the basic K-rich N-terminal and the acidic DE-rich C-terminal. (B) Phylogenetic analysis of plant HMGB proteins showing that CsHMG belongs to group B HMGBs. (C) Western-blot detection of the recombinant 6xHis-CsHMG (∼22 kDa) made in bacteria compared to bands detected in citrus cell extracts with the expected molecular size for the endogenous CsHMG (∼16 kDa). The anti-CsHMG serum also cross-reacted with a band of similar size in the cell extracts of <i>A. thaliana</i> wild-type and heterozygous <i>hmg-b1</i> mutant. This band, which has the expected molecular weight for AtHMGB1 (∼18 kDa), is less pronounced in the heterozygous <i>hmgb-1</i> mutant, thus indicating that CsHMG is structurally related to AtHMGB1.</p

<i>Citrus sinensis</i> proteins identified as targets of PthA4 are homologous to nuclear factors involved in chromatin remodeling and repair, transcription regulation and mRNA stabilization/modification.

<p><i>Citrus sinensis</i> proteins identified as targets of PthA4 are homologous to nuclear factors involved in chromatin remodeling and repair, transcription regulation and mRNA stabilization/modification.</p