Tomato 14-3-3 Proteins Are Required for Xv3 Disease Resistance and Interact with a Subset of Xanthomonas euvesicatoria Effectors.

The 14-3-3 phospho-binding proteins with scaffolding activity play central roles in the regulation of enzymes and signaling complexes in eukaryotes. In plants, 14-3-3 isoforms are required for disease resistance and key targets of pathogen effectors. Here, we examined the requirement of the tomato (Solanum lycopersicum) 14-3-3 isoform (TFT) protein family for Xv3 disease resistance in response to the bacterial pathogen Xanthomonas euvesicatoria. In addition, we determined whether TFT proteins interact with the repertoire of X. euvesicatoria type III secretion effector proteins, including AvrXv3, the elicitor of Xv3 resistance. We show that multiple TFT contribute to Xv3 resistance. We also show that one or more TFT proteins physically interact with multiple effectors (AvrXv3, XopE1, XopE2, XopN, XopO, XopQ, and XopAU). Genetic analyses indicate that none of the identified effectors interfere with AvrXv3-elicited resistance into Xv3 tomato leaves; however, XopE1, XopE2, and XopO are required to suppress symptom development in susceptible tomato leaves. Phospho-peptide mapping revealed that XopE2 is phosphorylated at multiple residues in planta and residues T66, T131, and S334 are required for maximal binding to TFT10. Together, our data support the hypothesis that multiple TFT proteins are involved in immune signaling during X. euvesicatoria infection.

Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector XopN from binding to TFT1 attenuate Xanthomonas virulence.

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344-733) is sufficient to bind TFT1. Removal of amino acids 605-733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.

Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1.

XopN is a virulence factor from Xanthomonas campestris pathovar vesicatoria (Xcv) that is translocated into tomato (Solanum lycopersicum) leaf cells by the pathogen's type III secretion system. Xcv DeltaxopN mutants are impaired in growth and have reduced ability to elicit disease symptoms in susceptible tomato leaves. We show that XopN action in planta reduced pathogen-associated molecular pattern (PAMP)-induced gene expression and callose deposition in host tissue, indicating that XopN suppresses PAMP-triggered immune responses during Xcv infection. XopN is predicted to have irregular, alpha-helical repeats, suggesting multiple protein-protein interactions in planta. Consistent with this prediction, XopN interacted with the cytosolic domain of a Tomato Atypical Receptor-Like Kinase1 (TARK1) and four Tomato Fourteen-Three-Three isoforms (TFT1, TFT3, TFT5, and TFT6) in yeast. XopN/TARK1 and XopN/TFT1 interactions were confirmed in planta by bimolecular fluorescence complementation and pull-down analysis. Xcv DeltaxopN virulence defects were partially suppressed in transgenic tomato leaves with reduced TARK1 mRNA levels, indicating that TARK1 plays an important role in the outcome of Xcv-tomato interactions. These data provide the basis for a model in which XopN binds to TARK1 to interfere with TARK1-dependent signaling events triggered in response to Xcv infection.