Suppression of a specific branch of mitogen-activated protein kinase (MAPK) signaling by bacterial AvrRpt2-like cysteine proteases: Mechanistic elucidation and application for MAPK studies.
Plant Physiology
Final Report Abstract
Phytopathogenic bacteria, such as Pseudomonas syringae, have evolved diverse mechanisms to suppress plant defence responses. This includes the injection of so-called effector proteins into the host cytosol, one of which is AvrRpt2 – a cysteine-protease known to cleave the plant defence modulating protein, RIN4. We previously uncovered another (putative) virulence function of AvrRpt2, namely the specific suppression of one of the two known branches of pathogen-responsive mitogen-activated protein kinases (MAPKs). Since MAPKs have both positive and negative roles in defence regulation, we proposed that this represents a fine-tuning of plant signalling rather than an indiscriminate “shutting down” of all MAPKs. An important difference to RIN4 is that the AvrRpt2-targeted MAPKs (MPK4/MPK11) are not directly cleaved – although the AvrRpt2 protease activity is essential. Thus, our working hypothesis for elucidating the underlying mechanism is “other (non-RIN4) cleavage substrates of AvrRpt2 are responsible for the selective inhibition of MPK4/MPK11 activation”. One of the chosen strategy was to use a recently developed mass spectrometry-based approach (so-called TAILS proteomics) to identify the novel N-termini of AvrRpt2 substrates. Besides initial technical problems, the pilot experiments did not identify known substrates such as RIN4 or related members from the so-called RIN4/NOI (where NOI = nitrate-induced) family. In line with these results, we subsequently found that some cleaved NOIs are genuine substrates of the so-called N-end rule pathway but others, like RIN4, are degraded through other pathways, thus offering a possible explanation for the negative results from the preliminary TAILS data. Using an alternative strategy that entails screening an AvrRpt2-like protease (Bp-AvrRpt2) from the soil bacterium, Burkholderia pyroccinia, for MPK4/11 suppression and correlating this to disappearance of putative substrates containing the consensus cleavage sites, we could narrow down six members of the RIN4/NOI family to be potentially involved. Higher order noi-mutants and silencing lines have been created and will be tested in the future with respect to MPK4/MPK11 suppression phenotype. Hence, this work is still ongoing and is crucial for improving our understanding of AvrRpt2 functions, which is a key virulence factor in important crop pathogens such as the apple/pear fire blight pathogen, Erwinia amylovora. In the context of additional AvrRpt2 functions, we have unpublished observations of another potential AvrRpt2 effect on early plant signalling and the expertise from the current project could be successfully applied for this analysis.
Publications
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Two strategies of Pseudomonas syringae to avoid recognition of the HopQ1 effector in Nicotiana species. Frontiers in Plant Science 9: 978 (2018)
Zembek P, Danilecka A, Hoser R, Eschen-Lippold L, Benicka M, Grech-Baran M, Rymaszewski W, Barymow-Filoniuk I, Morgiewicz K, Kwiatkowski J, Piechocki M, Poznanski J, Lee J, Hennig J, Krzymowska M
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(2019). Differential N-end rule degradation of RIN4/NOI fragments generated by the AvrRpt2 effector protease. Plant Physiology, 180 (4): 2272-2289
Goslin K, Eschen-Lippold L, Naumann C, Linster E, Sorel M, Klecker M, de Marchi R, Kind A, Wirtz M, Lee J, Dissmeyer N, Graciet E
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pH effects on plant calcium fluxes: lessons from acidification-mediated calcium elevation induced by the γ-glutamyl-leucine dipeptide identified from Phytophthora infestans. Scientific Reports 9: 4733 (2019)
Westphal L, Strehmel N, Eschen-Lippold L, Bauer N, Westermann B, Rosahl S, Scheel D, Lee J
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(2020). A mutation in Asparagine-Linked Glycosylation 12 (ALG12) leads to receptor misglycosylation and attenuated responses to multiple microbial elicitors. FEBS letters
Trempel F, Eschen-Lippold L, Bauer N, Ranf S, Westphal L, Scheel D, Lee J
