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Endoplasmic reticulum-plasma membrane contact sites (ER-PM CSs) are evolutionarily conserved membrane domains found in all eukaryotes, where the ER closely interfaces with the PM. This short distance is achieved in plants through the action of tether proteins such as synaptotagmins (SYTs). Arabidopsis comprises five SYT members (SYT1-SYT5), but whether they possess overlapping or distinct biological functions remains elusive. SYT1, the best-characterized member, plays an essential role in the resistance to abiotic stress. This study reveals that the functionally redundant SYT1 and SYT3 genes, but not SYT5, are involved in salt and cold stress resistance. We also show that, unlike SYT5, SYT1 and SYT3 are not required for Pseudomonas syringae resistance. Since SYT1 and SYT5 interact in vivo via their SMP domains, the distinct functions of these proteins cannot be caused by differences in their localization. Interestingly, structural phylogenetic analysis indicates that the SYT1 and SYT5 clades emerged early in the evolution of land plants. We also show that the SYT1 and SYT5 clades exhibit different structural features in their SMP and Ca 2+  binding of their C2 domains, rationalizing their distinct biological roles., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Plants encode numerous intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize pathogen-derived effectors or their activity to activate defenses. miRNAs regulate NLR genes in many species, often triggering the production of phased siRNAs (phasiRNAs). Most such examples involve genes encoding NLRs carrying coiled-coil domains, although a few include genes encoding NLRs carrying a Toll/interleukin-1 domain (TNL). Here, we characterize the role of miR825-5p in Arabidopsis, using a combination of bioinformatics, transgenic plants with altered miRNA levels and/or reporters, small RNAs, and virulence assays. We demonstrate that miR825-5p down-regulates the TNL MIST1 by targeting for endonucleolytic cleavage the sequence coding for TIR2, a highly conserved amino acid motif, linked to a catalytic residue essential for immune function. miR825-5p acts as a negative regulator of basal resistance against Pseudomonas syringae. miR825-5p triggers the production from MIST1 of a large number of phasiRNAs that can mediate cleavage of both MIST1 and additional TNL gene transcripts, potentially acting as a regulatory hub. miR825-5p is expressed in unchallenged leaves and transcriptionally down-regulated in response to pathogen-associated molecular patterns (PAMPs). Our results show that miR825-5p, which is required for full expression of PAMP-triggered immunity, establishes a link between PAMP perception and expression of uncharacterized TNL genes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The Pseudomonas syringae type III secretion system translocates effector proteins into the host cell cytosol to suppress plant basal immunity. Effector HopZ1a suppresses local and systemic immunity triggered by pathogen-associated molecular patterns (PAMPs) and effectors, through target acetylation. HopZ1a has been shown to target several plant proteins, but none fully substantiates HopZ1a-associated immune suppression. Here, we investigate Arabidopsis thaliana mitogen-activated protein kinase kinases (MKKs) as potential targets, focusing on AtMKK7, a positive regulator of local and systemic immunity. We analyse HopZ1a interference with AtMKK7 by translocation of HopZ1a from bacteria inoculated into Arabidopsis expressing MKK7 from an inducible promoter. Reciprocal phenotypes are analysed on plants expressing a construct quenching MKK7 native expression. We analyse HopZ1a-MKK7 interaction by three independent methods, and the relevance of acetylation by in vitro kinase and in planta functional assays. We demonstrate the AtMKK7 contribution to immune signalling showing MKK7-dependent flg22-induced reactive oxygen species (ROS) burst, MAP kinas (MAPK) activation and callose deposition, plus AvrRpt2-triggered MKK7-dependent signalling. Furthermore, we demonstrate HopZ1a suppression of all MKK7-dependent responses, HopZ1a-MKK7 interaction in planta and HopZ1a acetylation of MKK7 with a lysine required for full kinase activity. We demonstrate that HopZ1a targets AtMKK7 to suppress local and systemic plant immunity., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Many stimuli such as hormones and elicitors induce changes in intracellular calcium levels to integrate information and activate appropriate responses. The Ca(2+) signals are perceived by various Ca(2+) sensors, and calmodulin (CaM) is one of the best characterized in eukaryotes. Calmodulin-like (CML) proteins extend the Ca(2+) toolkit in plants; they share sequence similarity with the ubiquitous and highly conserved CaM but their roles at physiological and molecular levels are largely unknown. Knowledge of the contribution of Ca(2+) decoding proteins to plant immunity is emerging, and we report here data on Arabidopsis thaliana CML9, whose expression is rapidly induced by phytopathogenic bacteria, flagellin and salicylic acid. Using a reverse genetic approach, we present evidence that CML9 is involved in plant defence by modulating responses to bacterial strains of Pseudomonas syringae. Compared to wild-type plants, the later responses normally observed upon flagellin application are altered in knockout mutants and over-expressing transgenic lines. Collectively, using PAMP treatment and P. syringae strains, we have established that CML9 participates in plant innate immunity., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

*The Pseudomonas syringae pv syringae type III effector HopZ1a is a member of the HopZ effector family of cysteine-proteases that triggers immunity in Arabidopsis. This immunity is dependent on HopZ1a cysteine-protease activity, and independent of known resistance genes. We have previously shown that HopZ1a-triggered immunity is partially additive to that triggered by AvrRpt2. These partially additive effects could be caused by at least two mechanisms: their signalling pathways share a common element(s), or one effector interferes with the response triggered by the other. *Here, we investigate the molecular basis for the partially additive effect displayed by AvrRpt2- and HopZ1a-triggered immunities, by analysing competitive indices, hypersensitive response and symptom induction, PR-1 accumulation, expression of PR genes, and systemic acquired resistance (SAR) induction. *Partially additive effects between these defence responses require HopZ1a cysteine-protease activity, and also take place between HopZ1a and AvrRps4 or AvrRpm1-triggered responses. We establish that HopZ1a-triggered immunity is independent of salicylic acid (SA), EDS1, jasmonic acid (JA) and ethylene (ET)-dependent pathways, and show that HopZ1a suppresses the induction of PR-1 and PR-5 associated with P. syringae pv tomato (Pto)-triggered effector-triggered immunity (ETI)-like defences, AvrRpt2-triggered immunity, and Pto or Pto (avrRpt2) activation of SAR, and that suppression requires HopZ1a cysteine-protease activity. *Our results indicate that HopZ1a triggers an unusual resistance independent of known pathways and suppresses SA and EDS1-dependent resistance.

In recent years, many efforts have been directed towards the identification of new type III-secreted effectors, and the completion of the secretomes of several Pseudomonas syringae pathovars. Several functional and bioinformatic screenings have been used to search for candidates, which have been tested for translocation into the plant cell, an essential criterion for the identification of new type III effector proteins. The most common translocation assay is based on the use of DeltaAvrRpt2 as a reporter. When fused to a type III effector protein, DeltaAvrRpt2 is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 resistance protein. This approach has been used widely and has allowed the identification of a considerable number of new effectors in a fast and convenient manner. However, as the hypersensitive response is a semi-quantitative assay, and the conditions do not resemble those occurring in nature, effectors with low expression or translocation efficiency could fail to translocate sufficient DeltaAvrRpt2 to trigger a clear hypersensitive response. In keeping with these limitations, this test has failed to detect some true effectors that have been confirmed as such by other means. In order to increase the sensitivity of this method, we have developed a modification of the DeltaAvrRpt2-based translocation assay using a competitive index in mixed infection to monitor the limitation of growth associated with the induction of the hypersensitive response. We have tested several effector candidates from P. syringae pv. phaseolicola and other P. syringae pathovars, and have compared the results obtained by our competitive index translocation assay with those obtained by standard hypersensitive response assays. We have identified six type III secretion system-translocated proteins using this approach, five of which failed to be identified by hypersensitive response assays. In addition, we have analysed the defence response triggered by one of these effectors using competitive index assays.