Hydathode immunity against the vascular pathogen Xanthomonas campestris pv. campestris by the Arabidopsis CNL-type receptor SUT1

  1. Nanne W. Taks
  2. Marieke van Hulten
  3. Jeroen A. van Splunter-Berg
  4. Sayantani Chatterjee
  5. Misha Paauw
  6. Sebastian Pfeilmeier
  7. Harrold A. van den Burg

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Abstract

Bacterial plant pathogens exploit natural openings, such as pores or wounds, to enter the plant interior and cause disease. Plants actively guard these openings through defense mechanisms that have been described extensively for stomates, the most common points of entry. However, bacteria from the genus Xanthomonas have specialized in that they enter their host via hydathodes—a poorly studied organ at the leaf margin involved in guttation. While hydathodes can mount an effective immune response against bacteria, a dedicated perception mechanism still needs to be discovered. To identify a hydathode-specific immune receptor, we mapped a novel resistance gene against X. campestris pv. campestris (Xcc) in Arabidopsis using an inoculation procedure that promotes natural entry via hydathodes. Using Recombinant Inbred Lines (RILs) between susceptible accession Oy-0 and resistant Col-0, a QTL for resistance was identified on the right arm of Chromosome 5 in Col-0. Combining this finding with results of a genome-wide association analysis, a single candidate gene was fine-mapped that encoded a coiled-coil nucleotide-binding leucine-rich repeat (CNL) immune receptor protein called SUPPRESSOR OF TOPP4 1 (SUT1). Whereas the ZAR1 immune receptor acts in the vasculature against Xcc, we establish that SUT1 already restricts Xcc in hydathodes but is ineffective in the vasculature. In corroboration, we confirm promoter activity of SUT1 in the epithem tissue within hydathodes. Altogether, we provide evidence for an NLR that confers hydathode-specific resistance in Arabidopsis against infection by Xcc.

Author summary

Black rot disease, caused by the bacterial pathogen Xanthomonas campestris pv. campestris (Xcc), is an economically relevant disease of cabbage crops. Xcc is rather unique in that it enters the plant interior through specialized organs at the edge of the leaf. These structures called hydathodes contain water pores and are involved in leaf water regulation. Although we know that hydathodes can mount an immune response against these bacteria, specific immune receptors still need to be discovered. In our search for hydathode resistance mechanisms, we use the model plant Arabidopsis thaliana to identify genetic targets that could be translated to cabbage breeding practices. Here, by screening large populations of genetically diverse Arabidopsis plants, we could pinpoint a genetic locus that is involved in hydathode resistance. On this locus, we identified a gene, SUT1 , that confers resistance against Xcc, restricting early hydathode colonization by the bacteria and reducing subsequent disease symptoms. Interestingly, this resistance is ineffective in later stages of infection when the bacteria colonize the plant vascular system. Therefore, this study provides new insights in hydathode-specific resistance and opens doors for more research on these tissue- or organ-specific resistance mechanisms in plants.

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    Overview

    This paper by Taks et al. focuses on the identification and characterization of the hydathode-specific nucleotide-binding leucine-rich repeat (NLR) immune receptor, SUT1, in Arabidopsis thaliana and its role in conferring resistance against the vascular bacterial pathogen Xanthomonas campestris pv. campestris (Xcc). The authors aim to expand our understanding of tissue-specific resistance, which is crucial for developing durable resistance strategies for specialized vascular-colonizing pathogens. Given that Xanthomonas spp. can enter the xylem through hydathode colonization following guttation, this specific tissue is an important battle ground for studying bacterial-plant interactions.

    This work follows up on previous research which found that effector-triggered immunity (ETI) operates in hydathodes, as Xcc8004 multiplication is inhibited in the A. thaliana Col-0 accession by the action of the intracellular NLR immune complex of HOPZ-Activated Resistance 1 (ZAR1) and Resistance-Related Kinase 1 (RKS1) (Cerutti et al. 2017). ZAR1 detects the Xcc effector XopAC to elicit an immune response (Wang et al. 2015). Despite these finding, Xcc8004 DxopAC is still unable to colonize hydathodes, indicating another receptor may be involved (Paauw et al. 2023). The authors used a combination of recombinant inbred lines (RILs) between the susceptible accession Oy-0 and the resistant accession Col-0 to identify QTLs followed by Genome-wide association study (GWAS) to identify the candidate R-gene SUT1. This study reveals that SUT1 is mainly expressed in epithem cells within hydathodes to mediate effective resistance against XccDxopAC in hydathodes but not the vasculature. More specifically, the authors use sut1 T-DNA insertion lines to demonstrate reduced bacterial colonization and leaf chlorosis at the hydathodes after guttation inoculation, but no change in leaf chlorosis or bacterial progression after clip inoculation.

    The paper highlights the potential of using SUT1 as a candidate for developing engineered resistance in Brassica crops to ensure durable resistance. This gene makes for an excellent candidate for R-gene stacking in order to protect crops against Xanthomonas bacterial diseases.

    General Comments

    We enjoyed reading this paper, but we can only provide feedback on the data that we have access to. Unfortunately, we were unable to access the supplementary materials, so we only discuss the data that is presented in the main document.

    The authors did a really good job explaining the limitations of the study in which several QTLs were discovered from the RIL experiments, thus the observed resistance phenotype, that SUT1 contributes to, is quantitative. In fact, the sut1 T-DNA lines could not rescue the resistance phenotype to WT Col-0 levels indicating several genes are involved in hydathode resistance. We think that complementation of these lines through stable expression of SUT1 would provide stronger evidence supporting its role in bacterial restriction in these tissues. Additionally, some of the experiments could have used additional positive/negative controls which we expand upon further in the results section.

    Introduction:

    We think that the paper would benefit from further discussions on SUT1 in the context of other NLRs. More specifically, SUT1 has been previously described as a coiled-coil NLR (CNL) (Yan et al. 2019), yet it has only been described in context with another CNL ZAR1 in this paper. These two CNLs are very different in that SUT1 is a G10/GA CNL-type receptor and recent phylogenies show that it is quite divergent from other CNLs, including ZAR1 (Sunil et al. 2023). We encourage the authors to refer to recent literature with NLR phylogeny and discuss these findings in the introduction and discussion. Additionally, the authors should refer to SUT1 as a CNL-type receptor.

    Results:

    Overall, the results reported are of high quality and logically structured. Below we have a few comments/suggestions based on the main figures.

    Figure 1 – Mapping of SUT1 as a candidate R-gene

    • What are the specific regions from the RIL and GWAS experiments where SUT1 was mapped based on the markers? Please provide these in the text and provide a map of the gene architecture for this region.  It would be good to know what other genes are found in the region where SUT1 is encoded.

    • Panel C - Put border around 'NA' white legend, as it is difficult to see

    • Panel D - label markers

    • Two other QTL peaks above the LOD threshold were identified in this panel but they are not discussed further in the text. Are NLRs or immunity-related genes encoded within these regions?

    Figure 2 – SUT1 contributes to early hydathode resistance

    • In Panels A/B) include sizes for the domains/exons.

    Figure 3 – SUTI is expressed in epithem cells within hydathodes

    • Line 278  - "SUT1 is active in hydathodes" The authors do not investigate the activity of SUT1 in this paragraph, so a more appropriate title for this section would be: "SUT1 is expressed in hydathodes".

    • Panel A - There are no controls for the GUS assay. Please include Col-0 as a negative control to ensure that this localized expression pattern is not due to over staining. Additionally, the hydathode-specific promoter, pPUP1, can be used as a positive control.

    • In line 284 they mention that GUS staining was done with T1 plants. How were these confirmed to express the construct? Could data from S6 (T3 lines) also be included for this?

    • Panel C - SUT1 is expressed in hydathodes and mesophyll cells under normal conditions. We are curious if their expression pattern changes or becomes elevated in the context of infection (spray inoculation vs mock).

    Figure 4 – SUT1 resistance is ineffective when hydathodes are bypassed

    • Panel C - Although SUT1 acts specifically in hydathodes, Oy-0 has significantly higher bacterial progression through the vasculature in these assays. These results are not discussed or mentioned in this section.

    • ZAR1 is a good positive control in this clip inoculation assay testing vasculature colonization, it should be included in Figure 4 and not placed in Supplemental Figure 7.

    Discussion

    The discussion is well written. Below we have some comments on topics that we particularly enjoyed and topics that may need more context.

    See comments on introduction regarding SUT1 in the context of being a G10 CC-type NLR.

    The authors perform AlphaFold predictions on the Oy-0 and Col-0 SUT1 proteins, but they do not expand upon this in the discussion.

    The authors expression analysis of SUT1 shows that it is expressed in both hydathodes and mesophyll cells, so why is the resistance phenotype hydathode-specific? There are single-cell and tissue-specific RNASeq data available that can be used to determine/corroborate SUT1 tissue-specific expression. It would be nice to see if this data is supported by previous observations by Yagi et al. 2020 and Tang et al. 2023.

    Previous research from their lab demonstrated that ZAR1-independent hydathode colonization is dependent on the EDS1-PAD4-ADR1 pathway (Paauw et al. 2023). However, this pathway has been demonstrated to contribute mainly to TIR-NLR mediated resistance (Dongus and Parker 2021). An explanation of the potential mechanisms by which SUT1 triggers ETI could be proposed in the discussion.

    We particularly enjoy their hypothesis that SUT1 guards the TOPP4 phosphotase and therefore might be a component surveying ABA signalling manipulation at the hydathodes. Identification of the corresponding Xcc effector that trigger SUT1-mediated resistance and characterizing the role of the AvrE homolog XopAM in ABA signalling interference are exciting avenues for further investigation.

    Our conclusion

    This paper was a pleasure to read. These findings will be useful for uncovering mechanisms for hydathode resistance against Xcc.

    References

    Cerutti, A., Jauneau, A., Auriac, M-C, lauber, E., Martinez, Y., Chiarenza, S., Leonhardt, N., Bethomé, R., and Noël, L. D. 2017. Immunity at caulifower hydathodes controls systemic infection by Xanthomonas campestris pv campestris. Plant Physiology. 174(2):700-716.

    Dongus, J. A. and Parker, J. E. 2021. EDS1 signalling: At the nexus of intracellular and surface receptor immunity. Current Opinion in Plant Biology. 62:102039.

    Paauw, M., van Hulten, M., Chatterjee, S., Berg, J. A., Taks, N. W., Giesbers, M., Richard, M. M. S., van den Burg, H. A. 2023. Hydathode immunity protects the Arabidopsis leaf vasculature against colonization by bacterial pathogens. Current Biology. 33:697-710.

    Sunil, S., Beeh, S., Stöbbe, E., Fischer, K., Wilhelm, F., Meral, A., Paris, C., Teasdale, L., Jiang, Z., Aguilar Parras, E., Nürnberger, T., Weigel, D., Lozano-Duran, R., and El Kasmi, F. 2023. An atypical endomembrane localized CNL-type immune receptor with a conserved deletion in the N-terminal signaling domain function in cell death and immunity. BioRxiv. https://doi.org/10.1101/2023.09.04.556214.

    Tang B., feng, L., Hulin, M. T., Ding, P., and Ma, W. 2023. Cell-type-specific responses to fungal infection in plants revealed by single-cell transcriptomics. Cell Host & Microbe. 31(10):1732-1747.

    Wang, G., Roux, B., Feng, F., Guy, E., Li, L., Li, N., Zhang, X., Lautier, M., Jardinaud, M-F., Chabannes, M., Arlat, M., Chen, S., He, C., Noël, L. D., and Zhou, J-M. The decoy substrate of a pathogen effector and a pseudokinase specify pathogen-induced modified-self recognition and immunity in plants. Cell Host Microbe. 18(3):285-295.

    Yagi, H., Nagano, A. J., Kim, J., Tamura, K., Mochizuki, N., Nagatani, A., Matsushita, T., and Shimada, T. 2020. Fluorescent protein-based imaging and tissue-specific RNA-seq analysis of Arabidopsis hydathodes. Journal of Experimental Botany. 72(4):1260-1270.

    Yan, J., Liu, Y., Huang, X., Li, L., Hu, Z., Qin, Q., Yan, L., He, K., Wang, Y., and Hou, S. 2019. An unreported NB-LRR protein SUT1 is required for the autoimmune response mediated by type one protein phosphatase 4 mutation (topp4-1) in Arabidopsis. The Plant Journal. 100(2):357-373.

    Competing interests

    The authors declare that they have no competing interests.

  2. Version published to 10.1101/2024.06.20.599835v1 on bioRxiv