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Xanthomonas phaseoli pv. manihotis (Xpm) is the causal agent of cassava bacterial blight, the most important bacterial disease in this crop. There is a paucity of knowledge about the metabolism of Xanthomonas and its relevance in the pathogenic process, with the exception of the elucidation of the xanthan biosynthesis route. Here we report the reconstruction of the genome-scale model of Xpm metabolism and the insights it provides into plant-pathogen interactions. The model, iXpm1556, displayed 1,556 reactions, 1,527 compounds, and 890 genes. Metabolic maps of central amino acid and carbohydrate metabolism, as well as xanthan biosynthesis of Xpm, were reconstructed using Escher (https://escher.github.io/) to guide the curation process and for further analyses. The model was constrained using the RNA-seq data of a mutant of Xpm for quorum sensing (QS), and these data were used to construct context-specific models (CSMs) of the metabolism of the two strains (wild type and QS mutant). The CSMs and flux balance analysis were used to get insights into pathogenicity, xanthan biosynthesis, and QS mechanisms. Between the CSMs, 653 reactions were shared; unique reactions belong to purine, pyrimidine, and amino acid metabolism. Alternative objective functions were used to demonstrate a trade-off between xanthan biosynthesis and growth and the re-allocation of resources in the process of biosynthesis. Important features altered by QS included carbohydrate metabolism, NAD(P)(+) balance, and fatty acid elongation. In this work, we modeled the xanthan biosynthesis and the QS process and their impact on the metabolism of the bacterium. This model will be useful for researchers studying host-pathogen interactions and will provide insights into the mechanisms of infection used by this and other Xanthomonas species.
Due to their sessile lifestyle, plants are constantly exposed to pathogens and possess a multi-layered immune system that prevents infection. The first layer of immunity called pattern-triggered immunity (PTI), enables plants to recognise highly conserved molecules that are present in pathogens, resulting in immunity from non-adaptive pathogens. Adapted pathogens interfere with PTI, however the second layer of plant immunity can recognise these virulence factors resulting in a constant evolutionary battle between plant and pathogen. Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial leaf spot disease in tomato and pepper plants. Like many Gram-negative bacteria, Xcv possesses a type-III secretion system, which it uses to translocate type-III effectors (T3E) into plant cells. Xcv has over 30 T3Es that interfere with the immune response of the host and are important for successful infection. One such effector is the Xanthomonas outer protein M (XopM) that shows no similarity to any other known protein. Characterisation of XopM and its role in virulence was the focus of this work.
While screening a tobacco cDNA library for potential host target proteins, the vesicle-associated membrane protein (VAMP)-associated protein 1-2 like (VAP12) was identified. The interaction between XopM and VAP12 was confirmed in the model species Nicotiana benthamiana and Arabidopsis as well as in tomato, a Xcv host. As plants possess multiple VAP proteins, it was determined that the interaction of XopM and VAP is isoform specific.
It could be confirmed that the major sperm protein (MSP) domain of NtVAP12 is sufficient for binding XopM and that binding can be disrupted by substituting one amino acid (T47) within this domain. Most VAP interactors have at least one FFAT (two phenylalanines [FF] in an acidic tract) related motif, screening the amino acid sequence of XopM showed that XopM has two FFAT-related motifs. Substitution of the second residue of each FFAT motif (Y61/F91) disrupts NtVAP12 binding, suggesting that these motifs cooperatively mediate this interaction. Structural modelling using AlphaFold further confirmed that the unstructured N-terminus of XopM binds NtVAP12 at its MSP domain, which was further confirmed by the generation of truncated XopM variants.
Infection of pepper leaves, with a XopM deficient Xcv strain did not result in a reduction of virulence in comparison to the Xcv wildtype, showing that the function of XopM during infection is redundant. Virus-induced gene silencing of NbVAP12 in N. benthamiana plants also did not affect Xcv virulence, which further indicated that interaction with VAP12 is also non-essential for Xcv virulence. Despite such findings, ectopic expression of wildtype XopM and XopMY61A/F91A in transgenic Arabidopsis seedlings enhanced the growth of a non-pathogenic Pseudomonas syringae pv. tomato (Pst) DC3000 strain. XopM was found to interfere with the PTI response allowing Pst growth independent of its binding to VAP. Furthermore, transiently expressed XopM could suppress reactive oxygen species (ROS; one of the earliest PTI responses) production in N. benthamiana leaves. The FFAT double mutant XopMY61A/F91A as well as the C-terminal truncation variant XopM106-519 could still suppress the ROS response while the N-terminal variant XopM1-105 did not. Suppression of ROS production is therefore independent of VAP binding. In addition, tagging the C-terminal variant of XopM with a nuclear localisation signal (NLS; NLS-XopM106-519) resulted in significantly higher ROS production than the membrane localising XopM106-519 variant, indicating that XopM-induced ROS suppression is localisation dependent.
To further characterise XopM, mass spectrometry techniques were used to identify post-translational modifications (PTM) and potential interaction partners. PTM analysis revealed that XopM contains up to 21 phosphorylation sites, which could influence VAP binding. Furthermore, proteins of the Rab family were identified as potential plant protein interaction partners. Rab proteins serve a multitude of functions including vesicle trafficking and have been previously identified as T3E host targets. Taking this into account, a model of virulence of XopM was proposed, with XopM anchoring itself to VAP proteins to potentially access plasma membrane associated proteins. XopM possibly interferes with vesicle trafficking, which in turn suppresses ROS production through an unknown mechanism.
In this work it was shown that XopM targets VAP proteins. The data collected suggests that this T3E uses VAP12 to anchor itself into the right place to carry out its function. While more work is needed to determine how XopM contributes to virulence of Xcv, this study sheds light onto how adapted pathogens overcome the immune response of their hosts. It is hoped that such knowledge will contribute to the development of crops resistant to Xcv in the future.
In eukaryotes, regulated protein turnover is required during many cellular processes, including defense against pathogens. Ubiquitination and degradation of ubiquitinated proteins via the ubiquitin proteasome system (UPS) is the main pathway for the turnover of intracellular proteins in eukaryotes. The extensive utilization of the UPS in host cells makes it an ideal pivot for the manipulation of cellular processes by pathogens. Like many other Gram-negative bacteria, Xanthomonas species secrete a suite of type-III effector proteins (T3Es) into their host cells to promote virulence. Some of these T3Es exploit the plant UPS to interfere with immunity. This review summarizes T3E examples from the genus Xanthomonas with a proven or suggested interaction with the host UPS or UPS-like systems and also discusses the apparent paradox that arises from the presence of T3Es that inhibit the UPS in general while others rely on its activity for their function.
XopJ is a Xanthomonas type III effector protein that promotes bacterial virulence on susceptible pepper plants through the inhibition of the host cell proteasome and a resultant suppression of salicylic acid (SA) - dependent defense responses. We show here that Nicotiana benthamiana leaves transiently expressing XopJ display hypersensitive response (HR) -like symptoms when exogenously treated with SA. This apparent avirulence function of XopJ was further dependent on effector myristoylation as well as on an intact catalytic triad, suggesting a requirement of its enzymatic activity for HR-like symptom elicitation. The ability of XopJ to cause a HR-like symptom development upon SA treatment was lost upon silencing of SGT1 and NDR1, respectively, but was independent of EDS1 silencing, suggesting that XopJ is recognized by an R protein of the CC-NBS-LRR class. Furthermore, silencing of NPR1 abolished the elicitation of HR-like symptoms in XopJ expressing leaves after SA application. Measurement of the proteasome activity indicated that proteasome inhibition by XopJ was alleviated in the presence of SA, an effect that was not observed in NPR1 silenced plants. Our results suggest that XopJ - triggered HR-like symptoms are closely related to the virulence function of the effector and that XopJ follows a two-signal model in order to elicit a response in the non-host plant N. benthamiana.
XopJ is a Xanthomonas type III effector protein that promotes bacterial virulence on susceptible pepper plants through the inhibition of the host cell proteasome and a resultant suppression of salicylic acid (SA) - dependent defense responses. We show here that Nicotiana benthamiana leaves transiently expressing XopJ display hypersensitive response (HR) -like symptoms when exogenously treated with SA. This apparent avirulence function of XopJ was further dependent on effector myristoylation as well as on an intact catalytic triad, suggesting a requirement of its enzymatic activity for HR-like symptom elicitation. The ability of XopJ to cause a HR-like symptom development upon SA treatment was lost upon silencing of SGT1 and NDR1, respectively, but was independent of EDS1 silencing, suggesting that XopJ is recognized by an R protein of the CC-NBS-LRR class. Furthermore, silencing of NPR1 abolished the elicitation of HR-like symptoms in XopJ expressing leaves after SA application. Measurement of the proteasome activity indicated that proteasome inhibition by XopJ was alleviated in the presence of SA, an effect that was not observed in NPR1 silenced plants. Our results suggest that XopJ - triggered HR-like symptoms are closely related to the virulence function of the effector and that XopJ follows a two-signal model in order to elicit a response in the non-host plant N. benthamiana.