Studying the Plant Cell of pepper, revealing that the Xanthomonas type III effector XopS regulates the defense response and stomatal immunity of pepper

On February 2, 2022, the research team of Frederik Börnke of the Leibniz Institute of Vegetable and Ornamental Crops in Germany and the Institute of Biochemistry and Biology of the University of Potsdam published an article entitled "The Xanthomonas type-III effector XopS" in the internationally renowned journal The Plant Cell.
"stabilizes CaWRKY40a to regulate defense responses and stomatal immunity in pepper (Capsicum annuum)" research paper
.

This study found that virus-induced silencing of the WRKY40 gene in pepper enhances plant tolerance to infection by Xanthomonas campestris pv.
Vesicatoria (Xcv), and that the Xanthomonas effector protein XopS is Inhibition of defense gene expression enhances disease susceptibility, revealing its importance in suppressing stomatal immunity
.

https://doi.
org/10.
1093/plcell/koac032 As a key part of plant immunity, cells attacked by pathogens undergo rapid transcriptional reprogramming to minimize virulence
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Many bacterial plant pathogens use type III effector (T3E) proteins to interfere with plant defense responses, such as transcriptional reprogramming described above
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To investigate whether XopS affects pre-invasion defense responses in pepper, sensitive pepper plants were inoculated with a bacterial suspension of 1 × 108 CFU/mL
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At 7 dpi, the population density of XcvΔxopS bacteria was significantly lower than that of wild-type bacteria (Figure 1A)
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Growth of the XcvΔxopS strain was fully restored to wild-type levels by complementation of ectopic XopS expression
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It was observed by pressure infiltration that submerged inoculation with XcvΔxopS strain reduced the severity of infection, resulting in higher chlorophyll content in leaves, compared to leaves infected with wild-type Xcv or XcvΔxopS (XopS-HA) (Fig.
1B and C)
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Pepper leaves incubated with wild-type Xcv for 2 hours had no effect on stomatal pore size relative to mock-treated leaves (Fig.
1D)
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However, regardless of whether the leaves were treated with the XcvΔxopS strain or the XcvΔhrpF strain, the stomata of the leaves were significantly closed due to the inability to deliver T3Es to the host cells, while the XcvΔxopS (XopS-HA) strain was able to keep the stomata open (Fig.
1D)
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Therefore, the inhibitory effect of Xcv on stomatal closure is dependent on T3E, and XopS is the main contributor to this effect
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Figure 1.
XopS is required for full virulence of Xcv in susceptible pepper plants.
Next, the authors found that XopS-GFP interferes with the MTI response after Arabidopsis invasion
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Interestingly, the two transgenic lines with the highest XopS-GFP expression levels (lines 4 and 5) developed chlorotic symptoms about 5 days after β-estradiol treatment, independent of bacterial inoculation (Fig.
2B)
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This phenotype is reminiscent of T3E-expressing plants with an activated JA response
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Therefore, the authors analyzed the expression of JA marker genes induced early in the JA response in a representative transgenic line
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It was found that the expression of JA marker genes AtMYC2 and AtJAZ10 was significantly increased in XopS-GFP plants after 4 h of β-estradiol treatment (Fig.
2C)
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This is consistent with the increased JA levels in Arabidopsis plants expressing XopS-GFP 24 h after β-estradiol induction (Fig.
2D)
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Furthermore, inducible expression of XopS-GFP in Arabidopsis prevented stomatal closure after flg22 treatment (Fig.
2E)
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Therefore, the authors hypothesized that XopS functions similarly to COR in promoting bacterial susceptibility by inducing the JA response, which is less virulent when surface inoculated with Arabidopsis plants
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Figure 2 Inducible expression of XopS-GFP in transgenic Arabidopsis lines interferes with MTI and triggers JA signaling.
By screening a yeast two-hybrid (Y2H) cDNA library from Nicotiana tabacum, the authors identified WRKY40a as interacting with XopS protein
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Direct interaction assays in yeast showed that, in addition to NtWRKY40, XopS was able to interact with WRKY40 proteins from Arabidopsis thaliana and A.
benthamiana as well as pepper WRKY40a proteins (Fig.
3)
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Figure 3.
XopS interacts with WRKY40 in different plants in yeast If XopS interacts with WRKY40 in plants, the authors conjecture that their subcellular localization patterns will overlap
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Therefore, using a green fluorescent protein (GFP) fusion protein transiently expressed in N.
benthamiana leaves, the subcellular localization of XopS was found to overlap with its potential target protein in the plant nucleus (Fig.
4)
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Furthermore, XopS interacts with WRKY40 in vitro and in vivo
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Fig.
4 Interaction of WRKY40 and XopS in plants and in vitro To investigate whether WRKY40s interacting with XopS can activate transcription, the authors performed a yeast transcriptional activation assay
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It was found that silencing of CaWRK40a resulted in a faster and/or stronger defense response at the transcriptional level, which was associated with enhanced Xcv resistance
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Next, the authors verified whether CaWRKY40a is involved in Xops-induced stomatal immune disturbance
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After incubating leaves of CaWRKY40a-silenced plants with different Xcv strains for 2 h, the stomatal pore size of leaves was significantly reduced (Fig.
5A and B)
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Bacterial proliferation of CaWRKY40a-silenced plants was reduced by approximately 2 logs after soaking inoculated with wild-type Xcv (Fig.
5C), indicating that CaWRKY40a silencing significantly increased resistance to surface-inoculated Xcv bacteria
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Figure 5 The ability of XopS to interfere with pepper stomatal immunity requires WRKY40.
The authors used the VIGS tool on the Sol Genomics website to select a 300 bp fragment of the NbWRKY40 coding sequence to amplify and insert into the pTRV2 silencing vector
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VIGS resulted in an approximately 90% reduction in WRKY40 gene expression compared to pTRV2 controls (Fig.
6A)
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To investigate whether there is off-target silencing of other WRKY40 homologous genes in VIGS plants, the authors examined the transcript levels of NbWRKY40a and NbWRKY40e and found that they were not affected (Fig.
6B)
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Furthermore, XopS attenuated the ability of stomatal closure in response to MAMP stimulation (Fig.
6C)
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The expression of XopS-GFP also had no significant effect on the stomatal pore size of plants with reduced WRKY40 expression (Fig.
6C)
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Figure 6 Virus-induced silencing of the WRKY40 gene affects stomatal closure in N.
benthamiana in response to MAMP stimulation In conclusion, the authors propose a working model of how XopS promotes bacterial virulence (Figure 7)
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In the future, it is necessary to study the biological mechanism of how XopS prevents the proteasomal degradation of WRKY40 and reveal its possible enzymatic activity
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Figure 7 A working model of how XopS promotes Xcv toxicity Original link: https://academic.
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