Abstract
Key message
NbWRKY22 and NbWRKY25 are required for full activation of bacteria-associated pattern- and effector-triggered immunity as well as for the response to other non-bacterial defense elicitors.
Abstract
Plants defend themselves against pathogens using a two-layered immune system. Pattern-triggered immunity (PTI) can be activated upon recognition of epitopes from flagellin including flg22. Pseudomonas syringae pv. tomato (Pst) delivers effector proteins into the plant cell to promote host susceptibility. However, some plants express resistance (R) proteins that recognize specific effectors leading to the activation of effector-triggered immunity (ETI). Resistant tomato lines such as Rio Grande-PtoR (RG-PtoR) recognize two Pst effectors, AvrPto and AvrPtoB, and activate ETI through the Pto/Prf protein complex. Using RNA-seq, we identified two tomato WRKY transcription factor genes, SlWRKY22 and SlWRKY25, whose expression is increased during Pst-induced ETI. Silencing of the WRKY25/22 orthologous genes in Nicotiana benthamiana led to a delay in programmed cell death normally associated with AvrPto recognition or several non-bacterial effector/R protein pairs. An increase in disease symptoms was observed in silenced plants infiltrated with Pseudomonas syringae pv. tabaci expressing AvrPto or HopQ1-1. Expression of both tomato WRKY genes is also induced upon treatment with flg22 and callose deposition and cell death suppression assays in WRKY25/22-silenced N. benthamiana plants supported their involvement in PTI. Our results reveal an important role for two WRKYs as positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens.
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Acknowledgements
We thank Diane Dunham for helping with some of the plant inoculation experiments, Samantha Mainiero for technical advice and assistance and Diana Lauff for her assistance with microscopy. Mauro Bartolozzi and Santiago Martínez Alonso for plant care and Maria Gabriela Cano for helping with statistical analysis. This research was supported by Agencia Nacional de Promoción Científica y Técnica—Argentina (PICT2014-1589, HGR and PICT2017-0916, MAP); Consejo Nacional de Investigaciones Científicas y Técnicas—Argentina (PIP2014-0314, MAP and PUE INFIVE 2016-0110), and by National Science Foundation grant IOS-1546625 (GBM).
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RNR, GBM, MAP, and HGR designed the research. RNR and MAP performed the research. RNR, GBM, MAP., and HGR wrote the paper. All authors read and approved the manuscript.
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Table S1:
Details of the bacterial strains used in this study. Table S2: Description of primers used in this study. Table S3: Nucleotide sequences of fragments used for VIGS. Fig. S1. Transcript abundance of SlWRKY22, SlWRKY25, NbWRKY22a/b and NbWRKY25a/b. a Gene expression (RNA-seq) in tomato RG-PtoR leaves infiltrated with different Pst DC3000 mutant strains at 6 h after infiltration (units shown are RPKM). b Relative expression (qRT-PCR) in N. benthamiana-35S:Pto leaves challenged with different Pst DC3000 mutant strains to asses ETI activation. c Gene expression (RPKM) in RG-prf3 plants treated with 1 µM flg22, 30 minutes after infiltration. Bars represent the mean of three or four biological replicates with their corresponding standard deviation. Double asterisks indicate significant differences (corrected P value <0.01) with raw p-values corrected for multiple testing using the false discovery rate (Pombo et al. 2014; Pombo et al. 2017). Fig. S2. Phylogenetic analysis of group II-e of WRKY transcription factors using amino acid sequences. Green, black and red font and lines indicate Arabidopsis, N. benthamiana and tomato proteins, respectively. SlWRKY22 and SlWRKY25 and their N. benthamiana orthologous proteins are labeled next to the corresponding accession number. Tomato WRKY22 and WRKY25 are marked with a star. The PhyML method with a bootstrap of 100 replicates was used for the analysis. Fig. S3. Promoter analysis of tomato and N. benthamiana WRKY genes. Schematic representation of the promoter region (−1500 bp) of tomato (a) and N. benthamiana (b) genes. Arrows indicate translation start codon position and green triangles indicate the position of the W-boxes found with the analysis. Fig. S4. Analysis of the predicted targets of the VIGS constructs in N. benthamiana. VIGS tool (http://vigs.solgenomics.net/) output of SlWRKY25 (a), NbWRKY22 and NbWRKY25 (b) constructs. Graphical representation of siRNA (21 mers) from desired targets (blue) and possible off-targets (red). The best region for construct design suggested by the tool is shown in yellow. The expression of each gene (RPKM) based on previous RNA-seq data is shown on the right. Fig. S5. SlWRKY25-silenced N. benthamiana plants have compromised ETI. a Leaves of N. benthamiana plants silenced for Ec1, SlWRKY25 and SlPrf were syringe-infiltrated with a mix of Agrobacterium tumefaciens carrying Pto and AvrPto to elicit programmed cell death (PCD). The degree of PCD was monitored visually as explained in Materials and Methods. b N. benthamiana 35S:Pto plants were silenced and then syringe-infiltrated with 5 × 104 cfu/mL P. s. pv. tabaci expressing AvrPto or empty vector (EV). Percentage of the infiltrated leaf circles that developed disease symptoms is shown. Double and simple asterisks indicate significant differences compared with Ec1-silenced plants using Fisher’s exact test (**P <0.01; *P <0.05). Fig. S6. VIGS plants have a normal growth and development. Photograph of N. benthamiana plants silenced with the constructs shown, was taken four weeks after silencing. Fig. S7. Phylogenetic analysis of group II-e of N. benthamiana WRKYs using nucleotide sequences. Genes in this study are labeled next to the corresponding accession number. The possible off-targets of VIGS constructs used to target NbWRKY22ab and NbWRKY25ab are shown in red. Genes with accession numbers in grey have no expression (based on previous RNA-seq data). The PhyML method with a bootstrap of 100 replicates was used for the analysis. (PDF 1168 kb)
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Ramos, R.N., Martin, G.B., Pombo, M.A. et al. WRKY22 and WRKY25 transcription factors are positive regulators of defense responses in Nicotiana benthamiana. Plant Mol Biol 105, 65–82 (2021). https://doi.org/10.1007/s11103-020-01069-w
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DOI: https://doi.org/10.1007/s11103-020-01069-w