Synonymous sites for accessibility around microRNA binding sites in bacterial spot and speck disease resistance genes of tomato.

The major causes of mass tomato infections in both covered and open ground are agents of bacterial spot and bacterial speck diseases. MicroRNAs (miRNAs) are 16-21 nucleotides in length, non-coding RNAs that inhibit translation and trigger mRNA degradation. MiRNAs play a significant part in plant resistance to abiotic and biotic stresses by mediating gene regulation via post-transcriptional RNA silencing. In this study, we analyzed a collection of bacterial resistance genes of tomato and their binding sites for tomato miRNAs and Pseudomonas syringe pv. tomato miRNAs. Our study found that two genes, bacterial spot disease resistance gene (Bs4) and bacterial speck disease resistance gene (Prf), have a 7mer-m8 perfect seed match with miRNAs. Bs4 was targeted by one tomato miRNA (sly-miR9470-3p) and three Pseudomonas syringe pv. tomato miRNAs (PSTJ4_3p_27246, PSTJ4_3p_27246, and PSTJ4_3p_27246). Again, Prf gene was found to be targeted by two tomato miRNAs namely, sly-miR9469-5p and sly-miR9474-3p. The accessibility of the miRNA-target site and its flanking regions and the relationship between relative synonymous codon usage and tRNAs were compared. Strong access to miRNA targeting regions and decreased rate of translations suggested that miRNAs might be efficient in binding to their particular targets. We also found the existence of rare codons, which suggests that it could enhance miRNA targeting even more. The codon usage pattern analysis of the two genes revealed that both were AT-rich (Bs4 = 63.2%; Prf = 60.8%). We found a low codon usage bias in both genes, suggesting that selective restriction might regulate them. The silencing property of miRNAs would allow researchers to discover the involvement of plant miRNAs in pathogen invasion. However, the efficient validation of direct targets of miRNAs is an urgent need that might be highly beneficial in enhancing plant resistance to multiple pathogenic diseases.

WRKY22 and WRKY25 transcription factors are positive regulators of defense responses in Nicotiana benthamiana.

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. 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.

Mai1 Protein Acts Between Host Recognition of Pathogen Effectors and Mitogen-Activated Protein Kinase Signaling.

The molecular mechanisms acting between host recognition of pathogen effectors by nucleotide-binding leucine-rich repeat receptor (NLR) proteins and mitogen-activated protein kinase (MAPK) signaling cascades are unknown. MAPKKKα (M3Kα) activates MAPK signaling leading to programmed cell death (PCD) associated with NLR-triggered immunity. We identified a tomato M3Kα-interacting protein, SlMai1, that has 80% amino acid identity with Arabidopsis brassinosteroid kinase 1 (AtBsk1). SlMai1 has a protein kinase domain and a C-terminal tetratricopeptide repeat domain that interacts with the kinase domain of M3Kα. Virus-induced gene silencing of Mai1 homologs in Nicotiana benthamiana increased susceptibility to Pseudomonas syringae and compromised PCD induced by four NLR proteins. PCD was restored by expression of a synthetic SlMai1 gene that resists silencing. Expression of AtBsk1 did not restore PCD in Mai1-silenced plants, suggesting SlMai1 is functionally divergent from AtBsk1. PCD caused by overexpression of M3Kα or MKK2 was unaffected by Mai1 silencing, suggesting Mai1 acts upstream of these proteins. Coexpression of Mai1 with M3Kα in leaves enhanced MAPK phosphorylation and accelerated PCD. These findings suggest Mai1 is a molecular link acting between host recognition of pathogens and MAPK signaling.

Natural variation for unusual host responses and flagellin-mediated immunity against Pseudomonas syringae in genetically diverse tomato accessions.

The interaction between tomato and Pseudomonas syringae pv tomato (Pst) is a well-developed model for investigating the molecular basis of the plant immune system. There is extensive natural variation in Solanum lycopersicum (tomato) but it has not been fully leveraged to enhance our understanding of the tomato-Pst pathosystem. We screened 216 genetically diverse accessions of cultivated tomato and a wild tomato species for natural variation in their response to three strains of Pst. The host response to Pst was investigated using multiple Pst strains, tomato accessions with available genome sequences, reactive oxygen species (ROS) assays, reporter genes and bacterial population measurements. The screen uncovered a broad range of previously unseen host symptoms in response to Pst, and one of these, stem galls, was found to be simply inherited. The screen also identified tomato accessions that showed enhanced responses to flagellin in bacterial population assays and in ROS assays upon exposure to flagellin-derived peptides, flg22 and flgII-28. Reporter genes confirmed that the host responses were due primarily to pattern recognition receptor-triggered immunity. This study revealed extensive natural variation in tomato for susceptibility and resistance to Pst and will enable elucidation of the molecular mechanisms underlying these host responses.

Investigation on the Interaction of Pseudomonas syringae Effector AvrPto with AtRabE1d GTPase.

BACKGROUND: The tomato bacterial speck is a worldwide disease. It is caused by the infection of pathogenic Pseudomonas syringae pv. tomato which delivers the effector AvrPto into the host cells via the type III secretion system. AvrPto interacts with a Rab8 subfamily protein in the GTP-bound form and participates in the response to pathogen infection, but the pathogenic mechanism involved remains elusive. OBJECTIVES: The main objective of this study was to investigate on the interrelationship of AvrPto with AtRabE1d and Pto, which would allow us to have a deeper understanding of the pathogen mechanism of bacterial speck mediated by avirulent AvrPto and provides theoretic support for the prevention and cure of tomato bacterial speck disease. METHODS: AvrPto8-159 and AtRabE1d13-185Q74L proteins were expressed in Escherichia coli expression system, purified via nickel affinity chromatography and size exclusion chromatography, and identified by SDS-PAGE. The interaction of AvrPto8-159 with AtRabE1d13-185Q74L was confirmed in vitro based on the fluorescence resonance energy transfer. In addition, the affinity of AvrPto8-159 with AtRabE1d13-185Q74L:GTP was determined using the fluorescence polarization based equilibrium titration. The comparison of the complex structural model of AtRabE1d with AvrPto, which was docked and refined by Patchdock and FireDock softwares. RESULTS: AvrPto8-159 and AtRabE1d13-185Q74L can be expressed and purified well in E.coli. FRET between AvrPto8-159 with GFP-tag and mantGTP-load AtRabE1d13-185Q74L was observed slightly in vitro for the first time. On the other hand, deploying DsRed of AtRabE1d13-185Q74L as FRET partner with GFP, AvrPto was shown to interact with AtRabE1d more significantly with increasing concentrations of DsRed-AtRabE1d13-185Q74L. The equilibrium dissociation constant of AvrPto8-159 with AtRabE1d13-185Q74L:GTP was determined to be 13.5 µM. CONCLUSION: This work reports preparation and interaction of AvrPto8-159 with AtRabE1d13- 185Q74L. AvrPto8-159 exhibited medium affinity with AtRabE1d13-185Q74L based on the fluorescence polarization. From the structural model of the complex AvrPto:AtRabE1d, AvrPto interacted with AtRabE1d and Pto proteins with completely different biding sites.