NbWRKY22 and NbWRKY25 VIGS-silenced plants are impaired in PTI and ETI defense responses

POMBO, MARINA A.; ROSLI, HERNAN G.; RAMOS, ROMINA N.

Salta

Congreso; LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Plants defend themselves against pathogens using a two layered immune system. Pattern-triggered immunity (PTI) is activated upon recognition of microbe-associated molecular patterns (MAMPs). Particularly in tomato, the detection of bacterial flagellin relies on the perception of two MAMPs: flg22, that is recognized by the receptor flagellin sensing 2 (FLS2) and flgII-28, by flagellin sensing 3 (FLS3). Virulent bacteria as Pseudomonas syringae pv. tomato (Pst), the causal agent of tomato bacterial speck disease, can deliver effector proteins into the plant cell to promote susceptibility. However, some plants posses resistance (R) proteins that recognize specific effectors leading to the activation of effector-triggered immunity (ETI). Resistant tomatoes such as Río Grande-PtoR (RG-PtoR) recognize two Pst effectors (AvrPto and AvrPtoB) through Pto/Prf complex and then activate ETI.In a previous study, we identified two tomato WRKY transcription factors (TFs), SlWRKY22 and SlWRKY25, whose expression was induced specifically when ETI is activated at 6 hours post infiltration. Silencing of tomato WRKY ortholog genes in Nicotiana benthamiana (NbWRKYs) led to a delay in Pto/Prf-associated programmed cell death (PCD) development. Also, we observed an increase in disease symptoms in plants infiltrated with Pseudomonas syringae pv. tabaci (P. s. tabaci) expressing AvrPto. To confirm these results, we evaluated bacterial growth in leaf tissue and observed higher bacterial titer in plants silenced for NbWRKYs compared with control plants. On the other hand, we analyzed if these genes play a role in the recognition of another Pst effector, HopQ1-1 (effector recognized in N. benthamiana by Roq1 protein). Silenced plants infiltrated with P. s. tabaci expressing HopQ1-1 had more disease symptoms than the control ones, suggesting the participation of NbWRKYs in the recognition of more than one Pst effector.Arabidopsis thaliana WRKY22 (AtWRKY22, closest TF to SlWRKY22 and SlWRKY25), is implicated in early PTI activation. Using previously generated tomato transcriptomic data available at the Tomato Functional Genomic Database, we found that both tomato genes are induced when PTI is activated 30 minutes after treatment with flg22. To investigate the possible contribution of NbWRKYs to PTI, we performed a callose deposition assay and a cell death suppression assay (CDSA) in N. benthamiana silenced plants. Interestingly, both experiments confirmed the participation of NbWRKYs in PTI. Finally, we tested silencing efficiency and specificity of the different VIGS constructs used in our experiments using RT-qPCR. We observed a 90% reduction of transcript abundance using our VIGS-constructs in N. benthamiana plants compared with an Ec1-silenced negative control. Together, our results indicate the participation of both WRKY TFs as positive regulators of plant immunity.