Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding protein gene for canker formation

  1. Xinyu Chen
  2. Huasong Zou
  3. Tao Zhuo
  4. Wei Rou
  5. Wei Wu
  6. Xiaojing Fan

  • Curated by eLife

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    eLife assessment

    This valuable study provides new insight into potential subtle dynamics in effector biology. The data presented generally support the claims, but in some cases, significant controls are missing and so the overall work is currently incomplete. If the limitations can be addressed, this work should be of broad relevance for biologists interested in molecular plant-microbe interactions.

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Abstract

Xanthomonas citri subsp. citri ( Xcc ), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1 . This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620 , which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1.

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  1. Version published to 10.7554/elife.91684.3 on eLife
  2. Version published to 10.7554/elife.91684 on eLife
  3. Version published to 10.7554/elife.91684.2 on eLife
  4. eLife

    eLife assessment

    This valuable study provides new insight into potential subtle dynamics in effector biology. The data presented generally support the claims, but in some cases, significant controls are missing and so the overall work is currently incomplete. If the limitations can be addressed, this work should be of broad relevance for biologists interested in molecular plant-microbe interactions.

  5. eLife

    Reviewer #1 (Public Review):

    Previous Review:

    The authors have identified the predicted EBE of PthA4 in the promoter of Cs9g12620, which is induced by Xcc. The authors identified a homolog of Cs9g12620, which has variations in the promoter region. The authors show PthA4 suppresses Cs9g12620 promoter activity independent of the binding action. The authors also show that CsLOB1 binds to the promoter of Cs9g12620. Interestingly, the authors show that PthA4 interacts with CsLOB1 at protein level. Finally, it shows that Cs9g12620 is important for canker symptoms. Overall, this study has reported some interesting discoveries and the writing is generally well done. However, the discoveries are affected by the reliability of the data and some flaws of the experimental designs.

    Here are some major issues:

    The authors have demonstrated that Cs9g12620 contains the EBE of PthA4 in the promoter region, to show that PthA4 controls Cs9g12620, the authors need to compare to the wild type Xcc and pthA4 mutant for Cs9g12620 expression. The data in Figure 1 is not enough.

    The authors confirmed the interaction between PthA4 and the EBE in the promoter of Cs9g12620 using DNA electrophoretic mobility shift assay (EMSA). However, Fig. 2B is not convincing. The lane without GST-PthA4 also clearly showed mobility shift. For EMSA assay, the authors need also to include non-labeled probe as competitor to verify the specificity. The description of the EMSA in this paper suggests that it was not done properly. It is suggested the authors to redo this EMSA assay following the protocol: Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions PMID: 17703195.

    The authors also claimed that PthA4 suppresses the promote activity of Cs9g12620. The data is not convincing and also contradicts with their own data that overexpression of Cs9g12620 causes canker and silencing of it reduces canker considering PthA4 is required for canker development. The authors conducted the assays using transient expression of PthA4. It should be done with Xcc wild type, pthA4 mutant and negative control to inoculate citrus plants to check the expression of Cs9g12620.

    Fig. 6 AB is not convincing. There are no apparent differences. The variations shown in B is common in different wild type samples. It is suggested that the authors to conduct transgenic instead of transient overexpression.

    Gene silencing data needs more appropriate controls. Fig. D. seems to suggest canker symptoms actually happen for the RNAi treated. The authors need to make sure same amount of Xcc was used for both CTV empty vector and the RNAi. It is suggested a blink test is needed here.

    Comments on revised version:

    Point 1: Addressed well.

    Point 2: The EMSA was reconducted with adding unlabeled DNA, however, the results are still not convincing. Firstly, in fig.3D lane 5, with the absence of unlabeled DNA, the shifted bound signal wasn't reduced significantly. Secondly, still in fig.3D lane 5, the free labeled DNA probe at the bottom of the gel didn't increase. Which together mean that the unlabeled DNA was unable to compete with the labeled DNA and the "bound" shifted bands might not be true positive.

    Point 3: The authors didn't address the question clearly regarding the connection between the inhibition of Cs9g12620 promoter by PthA4 and the positive function of Cs9g12620 on citrus canker.

    Point 4: The comment was not addressed. Fig.7A and B are not convincing. Firstly, no evidence shows the expression of transiently expressed genes. Secondly, hard to tell the difference in 7A. Thirdly, since CsLOB1 positively regulates Cs9g12620, why expressing of CsLOB1 is unable to cause phenotype, while expression of PthA4 does?

    Point 5: addressed.

  6. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Reviewer #1:

    Point 1: The authors have demonstrated that Cs9g12620 contains the EBE of PthA4 in the promoter region, to show that PthA4 controls Cs9g12620, the authors need to compare to the wild type Xcc and pthA4 mutant for Cs9g12620 expression. The data in Figure 1 is not enough.

    The data in Figure 1 D and E show a pthA4 Tn5 insertion mutant Mxac126-80 and the expression level of Cs9g12620 in citrus inoculated with the pthA4 mutant.

    Point 2: The authors confirmed the interaction between PthA4 and the EBE in the promoter of Cs9g12620 using DNA electrophoretic mobility shift assay (EMSA). However, Figure 2B is not convincing. The lane without GST-PthA4 also clearly showed a mobility shift. For the EMSA assay, the authors need also to include a non-labeled probe as a competitor to verify the specificity. The description of the EMSA in this paper suggests that it was not done properly. It is suggested the authors redo this EMSA assay following the protocol: Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions PMID: 17703195.

    Thank you very much for your comments. We have re-conducted the EMSA analysis based on your suggestion. The DNA probe was labeled with Cy5, included a non-labeled probe as a competitor. (Figure 3 B and D; Figure 4B and E)

    Point 3: The authors also claimed that PthA4 suppresses the promote activity of Cs9g12620. The data is not convincing and also contradicts with their own data that overexpression of Cs9g12620 causes canker and silencing of it reduces canker considering PthA4 is required for canker development. The authors conducted the assays using transient expression of PthA4. It should be done with Xcc wild type, pthA4 mutant, and negative control to inoculate citrus plants to check the expression of Cs9g12620.

    We have detected Cs9g12620 expression in silencing citrus plants inoculated wild type Xcc 29-1. (Figure 7F)

    Point 4: Figure 6 AB is not convincing. There are no apparent differences. The variations shown in B are common in different wild-type samples. It is suggested that the authors conduct transgenic instead of transient overexpression.

    It has been proven that transient expression of PthA4 leads to canker-like phenotype, suggesting that this experiment is effective. However, it will be more confident if conduct transgenic plant overexpressing pthA4 and Cs9g12620. We’ll create the plants in our following research to confirm the phenotype.

    Point 5: Gene silencing data needs more appropriate controls. Figure D seems to suggest canker symptoms actually happen for the RNAi treated. The authors need to make sure the same amount of Xcc was used for both CTV empty vector and the RNAi. It is suggested a blink test is needed here.

    We used the same amount of Xcc to inoculate CTV empty vector and the RNAi. In either inoculation, the cultured Xcc cells were suspended in sterile distilled water to a final concentration of 108 CFU/mL (OD600 = 0.3).

    Point 6: Figure 1. Please draw a figure to clearly show the location of the EBE in the promoter of Cs9g12620, including the transcription start site, and translational start site.

    The EBE in Cs9g12620 promoter was indicated by underlined in Figure supplement 1. We did not sure about the translation start site, but the translation start site was labelled.

  7. Version published to 10.1101/2023.11.14.564028v2 on bioRxiv
  8. Version published to 10.7554/elife.91684.1 on eLife
  9. eLife

    eLife assessment

    This valuable study provides new insight into potential subtle dynamics in effector biology. The data presented generally support the claims, but in some cases controls are missing and so the overall work is currently incomplete. If the limitations can be addressed, this work should be of broad relevance for biologists interested in molecular plant-microbe interactions.

  10. eLife

    Reviewer #1 (Public Review):

    The authors have identified the predicted EBE of PthA4 in the promoter of Cs9g12620, which is induced by Xcc. The authors identified a homolog of Cs9g12620, which has variations in the promoter region. The authors show that PthA4 suppresses Cs9g12620 promoter activity independent of the binding action. The authors also show that CsLOB1 binds to the promoter of Cs9g12620. Interestingly, the authors show that PthA4 interacts with CsLOB1 at the protein level. Finally, it shows that Cs9g12620 is important for canker symptoms. Overall, this study has reported some interesting discoveries and the writing is generally well done. However, the discoveries are affected by the reliability of the data and some flaws in the experimental designs.

    Here are some major issues:
    The authors have demonstrated that Cs9g12620 contains the EBE of PthA4 in the promoter region, to show that PthA4 controls Cs9g12620, the authors need to compare to the wild type Xcc and pthA4 mutant for Cs9g12620 expression. The data in Figure 1 is not enough.

    The authors confirmed the interaction between PthA4 and the EBE in the promoter of Cs9g12620 using DNA electrophoretic mobility shift assay (EMSA). However, Figure 2B is not convincing. The lane without GST-PthA4 also clearly showed a mobility shift. For the EMSA assay, the authors need also to include a non-labeled probe as a competitor to verify the specificity. The description of the EMSA in this paper suggests that it was not done properly. It is suggested the authors redo this EMSA assay following the protocol: Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions PMID: 17703195.

    The authors also claimed that PthA4 suppresses the promote activity of Cs9g12620. The data is not convincing and also contradicts with their own data that overexpression of Cs9g12620 causes canker and silencing of it reduces canker considering PthA4 is required for canker development. The authors conducted the assays using transient expression of PthA4. It should be done with Xcc wild type, pthA4 mutant, and negative control to inoculate citrus plants to check the expression of Cs9g12620.

    Figure 6 AB is not convincing. There are no apparent differences. The variations shown in B are common in different wild-type samples. It is suggested that the authors conduct transgenic instead of transient overexpression.

    Gene silencing data needs more appropriate controls. Figure D seems to suggest canker symptoms actually happen for the RNAi treated. The authors need to make sure the same amount of Xcc was used for both CTV empty vector and the RNAi. It is suggested a blink test is needed here.

  11. eLife

    Reviewer #2 (Public Review):

    The following submission titled "Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding gene for canker formation" by Chen et al. provides evidence that PthA4 binds to PCs9g12620 to downregulate expression potentially for citrus canker disease development. They tackle a relevant, complicated problem about the timing and regulation of an S gene expression and its relationship to disease development. Most often research stops at an S gene that is upregulated. This study aims to define the complexity of TAL effector family proteins beyond their standard activation role. Cs9g12620 encodes a putative carbohydrate-binding protein, and downregulation of this occurs via PthA4-CsLOB1 direct interaction. Silencing of Cs9g12620 leads to reduced virulence of X. citri, highlighting its importance as an S gene target from PthA4-mediated CsLOB1 induction. The authors also hypothesize that PthA4 represses the expression of Cs9g12620, and it seems to depend not on DNA binding by PthA4 but rather CsLOB1 interaction. This provides an interesting mode of action for a TAL effector, which typically is described as a transcription factor. An overall curiosity is that TAL effectors like PthA4 induce gene expression for virulence activity, but the authors do not probe this question with artificial TAL effectors or PthA4 variants to define the domains required for this activity. These tools, which are widely used in TAL effector research, could help determine what domain is responsible for this repression and if it is unique to PthA4 or a general TAL phenomenon. Work is further needed to also demonstrate the repressive role of PthA4 over time because it is not explicitly clear that the time-related suppression is directly attributed to the PthA4-CsLOB1 interactions.

    (1) The authors show that both WT but not WT expressing AvrXa7 induce Cs9g12620 and CsLOB1. They performed an adjacent supportive experiment comparing a Tn5-disrupted pthA4 to WT and saw a similar induction. Do the authors have a southern blot or genome sequence to show this is the true mutation? Have the authors complemented the Tn5 strain with pthA4 and an artificial TAL effector?

    (2) Figure 2 and "The expression of Cs9g12620 depends on pthA4 during Xcc infection" section: Overall I cannot determine the biological importance as written in the text about examining an ortholog of Cs9g12620 that is not expressed. The title of Figure 2 is: "Cs9g12620 and Cs9g12650 show different profiles of expression owing to the genetic variation in promoter." What is the biological importance of showing that there is promoter variation when the RNA-seq pointed to this target? This is unclear. Now, an interesting experiment would be to create an artificial TAL that activates the expression of Cs9g12650, which was, yes, not expressed in Nicotiana, but this wasn't examined in citrus and could be with an artificial TAL effector. Moreover, if this is about how something is not expressed, this seems out of place in the story before we arrive at the repression aspect of the narrative. Is the lack of expression a typical state of this gene family and do TAL effectors induce this for virulence? Is it also possible that RT alone isn't sensitive enough to detect relevant Cs9g12650 expression? Could the authors rather build on their RNAseq data or maybe use qPCR, a more sensitive approach, to see if this gene is expressed. Overall, this seems like a non-issue still because it isn't clear why this is important to support their narrative. Finally "2 μg of total RNA extracted" seems to be an extremely high input for RT. In summary here, it would be nice to see the hypothesis they tested and how it supports their overall aim because this is unclear.

    (3) Figure 3C: The authors should include a 35S::GUS + 35S::pthA4 control. This control is missing to show that the suppression is not due to overexpressing the two proteins simultaneously.

    (4) Figure 3E&G are just the same but rotated. Please include a separate replicate as this would be more beneficial to examine. With this and concerns on some of the reporting, the raw data and images should be included as supplemental for each replicate and detailed as if they are a regular figure.

    (5) Figure 3G: What is low and high? There are quantifiable values (e.g. RLU) here that correspond to the intensity of the figure legend. There should be a water/buffer infiltrated control.

    (6) Figure 3F: The Y1H data demonstrate that PCs9g12620 is bound by PthA4. The second panel for the gel mobility shift is however lacking a complementary treatment with PCs9g12620 WT. These gel mobility shift assays are always relative to something, and there is no comparison here unfortunately to other treatments. An example to follow as a model for formatting and experimental design could include as seen in Figure 5 by Duan et al. MPP (DOI:10.1111/mpp.12667). These should be performed as a single experiment not separated by panel D. A GST-Tag only should always be an additional control.

    (7) Figure 4: CsLOB1 activates Cs9g12620. Figure 4C: A reasonable control would be to include 35S::GUS and 35S::PthA4.

    (8) Figure 5F: The purpose of this experiment to show the multiplication over time and increase is not clear. It would be expected to see an increase in growth over time during susceptibility; so why was this documented?

    (9) Figure 5: Cs9g12620 expression decreases along with expansion and pectin esterase expression. How do we know that this is not a general downregulation of gene expression more broadly due to cell death or tissue deformation at 10 dpi? To test if this is also PthA4-specific, an experiment needed would be to test a specific pthA4 mutant rather than the TAL effectorless strain, which is already pretty weak a pathogen and does not trigger expression of any tested genes to wild-type levels to see if this is a general trend or specific to PthA4 activity. Finally, why are the color bars switched for time points 5 & 10 dpi for the effectorless strain? This is the finding that led them to suggest the repression. According to the rest of the figure, the gray and black are typically 5 and 10 dpi, respectively, but they seem to be switched to fit the narrative.

    (10) Figure 6 nicely documents the interaction between PthA4 and CsLOB1, but why did the authors not take the additional step to define what domains are required for PthA4 interaction? This is an important curiosity of what mediates this interaction. Was it the repeats or C- or N-terminus? Is this general to TAL effectors or precise to PthA4? This seems like the crux of the story especially since there is a TAL effector binding cited in the promoter.

    (11) Figure 7: RNAi-mediated silencing of Cs9g12620 demonstrates that this gene is a susceptibility target for X. citri as seen by colonization (E). First, the symptoms are not quite clear in A, and the morphological changes are unclear. Are there additional images for these to showcase the difference reproducibly? They hypothesize that there is complexity in Cs9g12620 expression during infection as proposed in Figure 8. It seems pretty important to perturb this in the opposite direction with artificial TAL effectors that either target a) Cs9g12620 for induction and b) CsLOB1 in a 049E background. One would hypothesize that this would not allow for the CsLOB1 interaction because they demonstrate this is PthA4-specific and therefore Cs9g12620 expression would not decrease while CsLOB1 is induced.

    (12) Figure 8: It is unclear if this is an appropriate model. The impact of CsLOB1-PthA4 interaction is depicted as a late phenomenon based on Cs9g12620 expression. However, it is not clear from their data that the CsLOB1-PthA4 interaction does not happen at the early stages of infection. This is not defined by their experiments proposed. As mentioned above, an overall concern is that the authors do not test variants of PthA4 or domains that could examine specifically what permits this suppression. Is this a general TAL effector structure-mediated phenomenon or is it something unique about PthA4 in this family? Does it require both DNA binding and interaction with CsLOB1?

  12. Version published to 10.1101/2023.11.14.564028v1 on bioRxiv