Pepper pathogens: XopS pumps the brakes on the pepper immune response

All around us plants and pathogens are locked in an eons-long arms race. Plants continuously evolve ways to detect microbe-associated molecular patterns (MAMPs) and develop MAMP-triggered immunity (MTI). Meanwhile, pathogens evolve ways to evade MTI pathways or use such pathways against the host plant. Uncovering the molecular mechanics of these conflicts promises to lead to pathogen-resistant crops without the need for toxic chemical pesticides (Brookes, 2021). The pepper plant Capsicum annuum is an economically valuable crop often devastated by bacteria from the genus Xanthomonas (Potnis et al., 2015). Previous studies identified Xanthomonas outer protein S (XopS) as a Type III effector from X. campestris pv. vesicatoria (Xcv) that is necessary for infection, but not pathogenicity (Schulze et al., 2012). Given that XopS has no obvious sequence similarity with other proteins, the field was left wondering what XopS does to facilitate infection. New work by Margot Raffeiner and colleagues (Raffeiner et al., 2022) details the molecular role XopS plays in the evolutionary arms-race between Xcv and C. annuum (see Figure).

Figure.

Xcv delivers XopS into the host cell, which stabilizes WRKY40. WRKY40 inhibits SA-dependent immunity, ultimately leading to interference of stomatal closure. With stomatal closure inhibited, Xcv is free to enter leaf tissue. Adapted from Raffeiner et al. (2022), Figure 12B.

To identify the host target of XopS, the authors searched for host proteins that interact with XopS using a yeast two-hybrid screen. From this screen, they identified the transcription factor WRKY40 and validated that XopS interacts with WRKY40 homologs from Arabidopsis thaliana, Nicotiana benthamiana, and C. annuum. Additionally, of all the WRKY transcription factors tested, the XopS-WRKY40 interaction was the strongest, suggesting a high level of specificity. Finally, in an in vitro pull-down assay, the authors confirmed that the XopS-WRKY40 interaction is direct and does not require additional factors. Xcv uses XopS to specifically target CaWRKY40 to affect host transcription. The authors then sought to assess how XopS helps Xcv infect the plant.

A plant under attack from a pathogen must shift resources from normal metabolism to processes focused on fighting the pathogen. The authors demonstrated that CaWRKY40 mRNA levels rapidly increase after Xcv infection, confirming that CaWRKY40 is indeed involved in the pathogen response. Curiously, silencing CaWRKY40 resulted in fewer infection symptoms. This suggests that CaWRKY40 normally represses MTI. While transcriptomic changes upon infection necessitate upregulating genes responsible for fighting the pathogen, so too is it necessary to upregulate genetic repressors. Not only are repressors required for a tunable, dynamic transcriptomic response, but they are also necessary to turn off MTI when the threat has passed. CaWRKY40 is part of that turning off process. To Xvc, CaWRKY40 is an exploitable linchpin in C. annuum’s MTI.

The authors turned to the big remaining question of how XopS affects CaWRKY40. In tobacco leaf-infiltration experiments, the authors demonstrated that CaWRKY40 is degraded by the proteasome. CaWRKY40 levels increased with prolonged treatment of the proteasome inhibitor MG132, and the authors detected ubiquitinated CaWRKY40. Coexpression of CaWRKY40 and XopS resulted in stabilization of CaWRKY40 similar to MG132 treatment of wild-type plants. This suggests that XopS stabilizes CaWRKY40, allowing it to continue to function as an inhibitor of the immune response (see Figure). Upon infection, Xcv uses the plant’s own immune response off-switch against the plant, inhibiting the plant’s ability to fight off the pathogen. Curiously, the authors were unable to detect specific de-ubiquitination by XopS, nor could they collect evidence that XopS inhibits the proteasome in general. These findings leave the door open for further study on how XopS stabilizes CaWRKY40.

Plants are under near-constant attack from pathogens and pathogens are under immense selective pressure to find ways around plant immune responses. The work presented in Raffeiner at al. shows just how creative evolution can be in host–pathogen interactions. By studying such relationships, we may not only gain insight into the unique processes involved, but also discover novel mechanisms of gene and protein regulation.