A plant cell death-inducing protein from litchi interacts with Peronophythora litchii pectate lyase and enhances plant resistance

Cell wall degrading enzymes, including pectate lyases (PeLs), released by plant pathogens, break down protective barriers and/or activate host immunity. The direct interactions between PeLs and plant immune-related proteins remain unclear. We identify two PeLs, PlPeL1 and PlPeL1-like, critical for full virulence of Peronophythora litchii on litchi (Litchi chinensis). These proteins enhance plant susceptibility to oomycete pathogens in a PeL enzymatic activity-dependent manner. However, LcPIP1, a plant immune regulator secreted by litchi, binds to PlPeL1/PlPeL1-like, and attenuates PlPeL1/PlPeL1-like induced plant susceptibility to Phytophthora capsici. LcPIP1 also induces cell death and various immune responses in Nicotiana benthamiana. Conserved in plants, LcPIP1 homologs bear a conserved “VDMASG” motif and exhibit immunity-inducing activity. Furthermore, SERK3 interacts with LcPIP1 and is required for LcPIP1-induced cell death. NbPIP1 participates in immune responses triggered by the PAMP protein INF1. In summary, our study reveals the dual roles of PlPeL1/PlPeL1-like in plant-pathogen interactions: enhancing pathogen virulence through PeL enzymatic activity while also being targeted by LcPIP1, thus enhancing plant immunity.

The manuscript by Li et al describes the identification of two putative pectate lyases from the oomycete Peronophythora litchi (PlPeLs) whose function is required for virulence, as well as the identification of LcPIP1, a protein from litchi that interacts with both PlPeLs and that induces cell death when overexpressed in N. benthamiana.The authors go on to show that orthologues of LcPIP1 also induce cell death in N. benthamiana, and report that LcPIP1 interacts with BAK1/SERK3, and that the latter is required for LcPIP1-mediated cell death induction.The authors propose a model where LcPIP1 will be a positive regulator of immune responses that would interact with SERK3.The manuscript contains a very important amount of work.Some experiments, like the characterization of P. litchii mutants are of high standard, clear and support the claims, whilst other experiments raise some questions.The identification of LcPIP1 and of its orthologues in other plant species is noteworthy and suggests that the findings can be of general interest in the field of plant pathogen interactions.However, given the importance of the claims, other methodologies and/or experiments might be necessary to sustain them.For example, instead of gene silencing experiments, using CRISPR/Cas9 editing of SERK3/BAK1 in N. benthamiana and/or A. thaliana mutants will shed light on the role of SERK3 in the interaction.The same could be done for NbPIP1 and AtPIP1.
One of my concerns with the manuscript is that the methods are very limited, lacking in detail and even absent for several experiments.This, other than not allowing other researchers reproducing the work, makes difficult understanding some parts of the manuscript and complicates the task of assessing if the results presented justify the claims.Two examples of absences and their consequences follow: -The material and methods section for the qRT-PCRs for plant genes is absent.In the qRT-PCR analysis presented in Figures 3a and 4, the values for CK (Fig 3a) and RFP (Fig 4) appear to be the same at all time points.This is surprising, particularly for Fig 4, because agroinfiltration should give some level of induction of PTI-related genes.In absence of the methods, one possible explanation would be that the authors calculated the relative expression of the genes for each time point using the control for each time point as a reference, instead of using the earliest time point for controls for all samples.If this was the case, this would be misleading concerning the timecourse of gene expression.This point needs to be clarified and corrected if required.
-The material and methods section for most of the cloning is absent.In the text the authors describe creating PlPeL-M2s mutants, which imply mutating 7 Asp to Ala.There is no description in the methods of how this was achieved (one could guess how this was done from the primer list provided in supplementary data, but it is not specified in the methods).The same applies to most of the cloning done.
Related to this, in several CO-IP experiments, the IP for the target protein shows several bands, some of them present at the same intensity as the target protein, which are not present in controls (see Figures 7 and Suppl 7).What could these proteins be and how do the authors rule out that the interaction does not take place with one of these proteins instead of with the target protein?Finally, in most cases the authors present their results as histograms with the means ± SE of independent biological replicates.I suggest adding the data points to the histograms in order to have a better view of the dispersion of the data points, and identifying the different repeats (alternatively use boxplots or other presentation that helps visualizing dispersion).Numbers of datapoints should be indicated for all experiments.I assume that the authors confirmed that their dataset fulfils the requirements for the statistical test used.

Abbreviate differentially Phytophthora and Peronophythora
Overall, since the authors have not demonstrated pectate lyase activity, it might be more appropriate using the terms putative or candidate pectate lyases.
Overall, the authors use lesion diameter to account for pathogen infection, but lesions are not always circular, in particular for experiments on litchi.Using lesion area would be more accurate.
Overall, the figure legends are limited.This is particularly true for the legends of the Supplementary Figures.P7, l 118-122.The claim that up-regulation of either PlPeL1 and PlPeL1-like upon knocking out the other supports functional redundancy should be played down.In Suppl Fig 1 the authors show that both genes are separated by approx.1100 bp.This proximity in the promoters for both genes, even if they are in opposite strands, could lead to a competition for expression in the WT, and thus to an increase of the expression of one of the genes when the other is knocked down.P7, l 130-131 and Suppl Fig 5 .In order to highlight the conservation of the Asp residues, it could be more interesting to present the alignment of PlPeLs with other PeLs from other species.Related to this, the sentence in l 130 needs to be developed.Define positive and negative controls in Figure 2a P11, l 208.The conserved motif VDMASG could be extended to VDMASGK/RYLW P11, l 209.It is not clear to me what is the contribution to the manuscript of the phylogenetic tree presented in Figure 5a.P11, l 209.Provide accession numbers for all proteins in Suppl Figure 9. P15, l 297.NbPIP1 is upregulated upon INF-1 mediated cell death (Figure 8d) as well as upon PlPeL1 expression (Suppl Figure 11).INF1 induces defense responses and PlPeLs suppresses defense responses.How does it fit with the model of NbPIP1 function?
As a non-native English speaker, I understand how frustrating this can be, but the manuscript would benefit from proof-reading by a native English speaker.
Reviewer #2 (Remarks to the Author): This MS describes the identification and characterisation of two secreted proteins (PlPel1 and PlLel-like proteins) from Peronophythora litchii and their target.
In short, the authors show that: 1-deletion of both genes in P. litchii results in a loss of virulence 2-Said Pl proteins interact with a small secreted protein (Pip1) in plants 3-LcPIP mediates cell death (orchestrated by a small and highly conserved motif) 4-Plant cell death resembles a PTI-type response that requires SERK3 and activate expression of known PTI responsive genes Based on these observations, the authors conclude that LcPIP is a cell death inducing peptide that interacts with two Pl effectors.
Overall, the work is presented nicely and the MS is of good written quality.Major comments are listed below: -It is not clear to me what the main message of the work is and how it is important.LcPIP induces cell death when over-expressed in plants and its silencing compromises immunity to virulent pathogens.This is an interesting observation (bolstered by interesting interaction data).Does this reduced immunity phenotype extend to non-pathogenic microbes?Reduction of PTI gene expression hints at a more general impairment, but this is not explored.That's where I think exploration would lead to exciting discoveries.
-The authors show that two Pl effectors bind Litchi (and Nb) PIP AND that disabling both effectors affect virulence.The significance of these observation is not clear.What is the consequence of PIP1 binding?Disabling of PIP1 function or PAMP perception?The authors will know that knocking out two effector coding genes could cause a virulence phenotype that has little to do with LcPIP1 signalling.Infection assays that feature knockout strains and PIP1 mutants would help address that.
It is possible that PIP1-mediated immunity is actively suppressed during infection and if so, understanding the mechanisms by which Pl disrupts its signalling would be of great interest.As it stands, however, there is not enough data to support a credible model that informs on the action of PIP1 and (possibly) Pl virulence factors.The proposed model reflect these knowledge gaps as it is fairly vague and throws up many questions.
Reviewer #3 (Remarks to the Author): This manuscript describes the involvement of pectate lyases (PlPeL1 and PlPeL1-like) from Peronopthora litchii in pathogenicity and a novel plant cell death-inducing protein, LcPIP1, from litchi.First, the authors created single or double deletion mutants of PlPeL genes and demonstrated that the double deletion mutant impaired the pathogenicity of P. litchi.Next, the authors investigated the interaction of PlPeLs and PIP1s.They demonstrated that the signal peptides and the VDMASG motif of LcPIP1 were essential to induce PCD in Nicotiana benthamiana.Moreover, they showed that the plant immune response by LcPIP/NbPIP was triggered via SERK3.The data presented in this manuscript is very interesting and the methods used are reasonable.The manuscript, however, has several critical issues.I would recommend this manuscript for publication in Nature Communications, provided the authors reasonably address the following points.
Line 91: Is the identity (99.78%) ranged from 1 to 460 amino acids?These two pel genes can be named as PlPeL1 and PlPeL2 because the C-terminal regions of the two proteins are quite different.That makes easier to distinguish them in the manuscript.The authors don't have to change the names but I strongly recommend.
Line 125: Was the full length of PlPel1-lilke protein expressed including the unique C-terminus?Line 142: Which means that the increase of the susceptibility was responsible for only the degradation of pectic substances (pectate)?Do PlPeL1M2 and -likeM2 contain signal peptides?Line 166: Have you conducted the expression assay of LcPIP1 using litchi inoculated with a single or double deletion mutants of Pel genes?Apparently, Pels up-regulate NbPIP1 in N. benthamiana from the results of Supplementary Fig. 11.Whether the expression of LcPIP1 is also regulated by Pels in litchi is intriguing.
Line 233: Single (PIP) and co-expressing (PIP/PeL) leaves showed the same lesion size, which raises the following question: PlPeL1 and -like have no effect on the infection of P. cspsici?I just wonder how the complex of LcPIP1/PlPeL1 is involved in pathogenicity and plant resistance.I understand that LcPIP1 indeed induced PCD in N. benthamiana from your data.Do you expect that PlPeLs from P. litchii have function to inhibit LcPIP1 from binding to SERK3 through interacting with each other in addition to degradation of pectic substances? or LcPIP is secreted to inhibit the enzymatic activity of PlPeLs in addition to the induction of immune systems?I understand that they have not completely proven yet in you work, but I believe it is necessary to describe your prediction at least.From the title of your paper, readers will imagine the effect given by the interaction between Pel and PIP toward pathogenicity and the plant immune systems.
Line 251: Do you mean that the susceptibility induced by PlPeL1 is different from that by PlPeL1-like in mechanisms?If NbPIP1 has higher ability to attenuate the susceptibility induced by PlPeL1, I just wonder why leaves expressing PlPeL1 or PlPeL1-like, expressing PIP1 as well, showed the same lesion size (Figure 1d-g and Figure 6ab).

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): The manuscript by Li et al describes the identification of two putative pectate lyases from the oomycete Peronophythora litchi (PlPeLs) whose function is required for virulence, as well as the identification of LcPIP1, a protein from litchi that interacts with both PlPeLs and that induces cell death when overexpressed in N. benthamiana.The authors go on to show that orthologues of LcPIP1 also induce cell death in N. benthamiana, and report that LcPIP1 interacts with BAK1/SERK3, and that the latter is required for LcPIP1-mediated cell death induction.The authors propose a model where LcPIP1 will be a positive regulator of immune responses that would interact with SERK3.The manuscript contains a very important amount of work.Some experiments, like the characterization of P. litchii mutants are of high standard, clear and support the claims, whilst other experiments raise some questions.The identification of LcPIP1 and of its orthologues in other plant species is noteworthy and suggests that the findings can be of general interest in the field of plant pathogen interactions.However, given the importance of the claims, other methodologies and/or experiments might be necessary to sustain them.For example, instead of gene silencing experiments, using CRISPR/Cas9 editing of SERK3/BAK1 in N. benthamiana and/or A. thaliana mutants will shed light on the role of SERK3 in the interaction.The same could be done for NbPIP1 and AtPIP1.
Re: We would like to express our gratitude to the reviewer for valuable time and thoughtful feedback, which has greatly contributed to the improvement of our manuscript.In the revised manuscript, we incorporated data from Arabidopsis thaliana and Nicotiana benthamiana mutants generated through CRISPR/Cas9 editing.
Our results showed that AtPIP1 also interacts with AtBAK1 (new Supplementary Fig. 13b) and loss of AtPIP1 led to higher susceptibility to pathogens (new Fig. 3g-i).
Deletion of NbBAK1 significantly reduced cell death and compromised the plant's resistance induced by LcPIP1 (new Fig. 6).These results made our results more solid and demonstrated that PIP1 and its homologs from different plants could positively contribute to plant immunity, which suggest that the findings can be of general interest in the field of plant pathogen interactions.
One of my concerns with the manuscript is that the methods are very limited, lacking in detail and even absent for several experiments.This, other than not allowing other researchers reproducing the work, makes difficult understanding some parts of the manuscript and complicates the task of assessing if the results presented justify the claims.Two examples of absences and their consequences follow: -The material and methods section for the qRT-PCRs for plant genes is absent.In the qRT-PCR analysis presented in Figures 3a and 4 In absence of the methods, one possible explanation would be that the authors calculated the relative expression of the genes for each time point using the control for each time point as a reference, instead of using the earliest time point for controls for all samples.If this was the case, this would be misleading concerning the time-course of gene expression.This point needs to be clarified and corrected if required.
-The material and methods section for most of the cloning is absent.In the text the authors describe creating PlPeL-M2s mutants, which imply mutating 7 Asp to Ala.There is no description in the methods of how this was achieved (one could guess how this was done from the primer list provided in supplementary data, but it is not specified in the methods).The same applies to most of the cloning done.
Re: We thank the reviewer for the comments, and we have taken steps to address these concerns in our revised manuscript.Specifically, we have revised the manuscript to include a more detailed description of the experimental methods utilized in the revised Methods section.Furthermore, we have revised our figures (new Fig. 5) and relative sentences for curate expression (figure legend of Fig. 5).Moreover, we have included additional details on the generation of amino acid mutants for PlPeL1, PlPeL1-like, and LcPIP1 in the ''Generation of amino acid mutants of PlPeL1, PlPeL1-like, and LcPIP1'' section of the Methods.
Another main concern has to do with the versions of the protein carrying or not a signal peptide (SP).In P8 and Figure 1 the authors claim that the PlPeL1 mutant lacking the SP (M1) lost its virulence function.This would make sense because PlPeL1 is meant to function in the extracellular space, and absence of SP means that the protein will not be secreted.However, on Suppl Fig 6, M1 is barely detectable compared to the WT, so it is difficult to conclude that this mutant lost its virulence function.The same applies to LcPIP1qSP: the fact that the protein is barely detectable YdZhcti Vaadl id YgVl V XdcXajh^dc VWdji i]Z gdaZ d[ i]Z NK ^c XZaa-death induction (Figure 3).
Re: We expressed full length of PlPeL1/PlPeL1-like with a lower agrobacterium concentration (OD600=0.0005),and the protein accumulation of PlPeL1/PlPeL1-like was still higher than that of M1 mutants (Author Response Figure 1, for review only).

Indeed, the loss of virulence in PlPeL1
& SP /PlPeL1-like & SP is very likely due to the low protein accumulation.Therefore, we have removed the results of leaves expressing PlPeL1 & SP or PlPeL1-like & SP inoculated with Ph. capsici.Meanwhile, we have renamed the mutant with amino acid replacement (7 Asp to Ala) as M1.

Author Response Figure 1 (For review only) Immunoblot analysis of PlPeL1
& SP and PlPeL1-like & SP expressed in N. benthamiana leaves.Total proteins were extracted from N. benthamiana leaves at 36 hpa.Red asterisks indicated protein bands of the correct size.Ponceau S staining of Rubisco was used to indicate loading quantity of protein samples.
This is important because, based on the model proposed for the authors in Figure 8, PlPeLs and LcPIP1 function in the extracellular space, but in reality it is not clear if PlPeLs is being secreted when transiently expressed in N. benthamiana.For LcPIP1 the authors show that it is partially secreted in yeast.Concerning its expression in N. benthamiana, looking at Figure 3d, the detected LcPIP1 protein appears bigger than LcPIP1qSP, suggesting that the signal peptide is still present in the protein.Because SP-mediated secretion involves cleavage of the SP, this would mean that most of the detected protein is still intracellular, which will be difficult to reconcile with the protein interacting with the extracellular part of SERK3.
Re: Thank you for your comments.In the revised manuscript, we also added new experiment to demonstrate that PlPeL1, PlPeL1-like, and LcPIP1 were secreted into apoplast.
First, we supplemented the western blot analysis of apoplastic fluid from N. benthamiana leaves agroinfiltrated with PlPeL1, PlPeL1-like, and LcPIP1.PlPeL1, PlPeL1-like, and LcPIP1 could be successfully detected in apoplastic fluid using immunological analysis (new Fig. 1b and new Fig.4f), indicating that three proteins were indeed secreted into the apoplastic space.
Second, we supplemented the experiment to validate the secretory function of the signal peptide of PlPeL1 and PlPeL1-like by yeast signal trap assay (new Fig. 1a), and results supported that the signal peptides of PlPeL1 and PlPeL1-like possess secretory function.
Third, it has been previously reported that PsXEG1 must be targeted to the extracellular space of N. benthamiana tissue to induce cell death (Ma et al., 2015).We noticed that expression of LcPIP1 SP -PsXEG1 {NK was able to induce cell death, whereas PsXEG1 {NK or LcPIP1 SP -RFP could not induce cell death in N. benthamiana (new Supplementary Fig. 11c).In addition, both LcPIP1 SP -PsXEG1 {NK and PsXEG1 could be detected as two isoforms (new Supplementary Fig. 11d).These results further confirmed that the signal peptide of LcPIP1 has the secretory function.
Fourth, SERK3 (BAK1) possesses both an extracellular domain (ECD) leucinerich repeat (LRR) and an intracellular cytoplasmic domain (CD) (Jaillais et al., 2011) (new Fig. 7d).In the revised manuscript, Co-IP assays showed that the extracellular domain of LcSERK3 interacted with LcPIP1, but not the cytoplasmic domain (new Fig. 7e).This result further confirms that LcPIP1 interacted with the extracellular part of LcSERK3.In figure 3d it is also noteworthy that the protein carrying the PR1-SP appears smaller than LcPIP, when it should be at least identical (if not bigger, based on the cartoon in Fig 3c).This could be explained if the PR1-SP was processed, but then the size should be the same as LcPIP1qSP, which is not.All this needs to be clarified.

References
Re: First of all, we apologize, as there might be unknown issues with the Agrobacterium tumefaciens strains carrying C-terminal HA-tagged PR1 SP -LcPIP1 {NK constructs.In response to this concern, we conducted a repeated Agrobacterium transformation for the three plasmids (pBin-LcPIP-HA, pBin-PR1 SP -LcPIP1 Z86 -HA, and pBin-LcPIP1 Z86 -HA) and subsequently infiltrated the resulting single colonies into N. benthamiana.Total proteins were then extracted from N. benthamiana leaves at 36 hpa.Our findings demonstrated that the protein sizes of LcPIP1-HA and PR1 SP -LcPIP1 {NK -HA were essentially similar, and both were larger than LcPIP1 {NK -HA (new Supplementary Fig. 11b, or Author Response Figure 2).Consistent with the previous results, expression of LcPIP1 or PR1 SP -LcPIP1 {NK induced cell death at 3 dpa, while LcPIP1 {NK could not induce cell death in N. benthamiana (new Fig. 4g).These experiments were repeated three times, and we consistently obtained similar results.To address this issue, we have rectified the problem in the blots and provided the blot photos in the Source data file for further inspection.
We speculate that LcPIP1 may undergo unknown post-translational modifications, resulting in an increase in the molecular weight of the protein.For example, both LcPIP1 SP -PsXEG1 {NK and PsXEG1 could be detected two isoforms, but not PsXEG1 {NK (new Supplementary Fig. 11d).Furthermore, we also noticed that the accumulation of the LcPIP1 {NK protein was lower.In N. benthamiana expressing either LcPIP1 or LcPIP1 {NK , the transcription levels of LcPIP1 were comparable (Author Response Figure 3a).Therefore, the discrepancy of protein accumulation may be attributed to the lack of proper localization instructions in LcPIP1 {NK , which could potentially have affected its stability and lead to its degradation or instability within the cell.
Considering the diminished accumulation of LcPIP1 {NK protein relative to LcPIP1, we investigated whether LcPIP1 could still induce cell death at lower protein levels.Our findings demonstrated that LcPIP1 retained the ability to induce cell death in N. benthamiana, even when its protein level was reduced (Author Response Figure 3b,c).187, 321-335 (2021).Liu, Z. et al.SRC2-1 is required in PcINF1-induced pepper immunity by acting as an interacting partner of PcINF1.J. Exp. Bot. 66, 3683-3698 (2015).

Author Response
Related to this, in several CO-IP experiments, the IP for the target protein shows several bands, some of them present at the same intensity as the target protein, which are not present in controls (Figures 7 and Suppl 7).What could these proteins be and how do the authors rule out that the interaction does not take place with one of these proteins instead of with the target protein?
Re: Thanks for the comments.Previous reports have indicated that that SERK3 (BAK1) undergoes Ca 2+ -dependent proteolytic cleavage in planta, which plays an essential role in BAK1-regulated plant immunity (Zhou et al., 2019).
In response to your concern, we have confirmed the interaction using RFPtagged PIP1s and HA-tagged LcSERK3, NbSERK3A, NbSERK3B, and AtBAK1 (new Fig. 7c).Combined with previous experiments, our results proved that LcPIP1 interact with LcSERK3/NbSERK3a in plant (new Fig. 7).
Regarding Suppl 7 (new Supplementary Fig. 6b), we confirmed the interaction between RFP-tagged LcPIP1 and HA-tagged PlPeL1/PlPeL1-like (Fig. 2b).Furthermore, GST pull-down have further verified that the target protein, rather than degraded protein, interacted with LcPIP1 (new Supplementary Fig. 6c).Finally, in most cases the authors present their results as histograms with the means ± SE of independent biological replicates.I suggest adding the data points to the histograms in order to have a better view of the dispersion of the data points, and identifying the different repeats (alternatively use boxplots or other presentation that helps visualizing dispersion).Numbers of datapoints should be indicated for all experiments.I assume that the authors confirmed that their dataset fulfils the requirements for the statistical test used.

References
Re: Thank you for your suggestions.We have added the data points to all the histograms.

Other suggestions
Abbreviate differentially Phytophthora and Peronophythora Re: Thanks for pointing this out.We have abbreviated them differentially as Phytophthora (Ph.) and Peronophythora (P.) Overall, since the authors have not demonstrated pectate lyase activity, it might be more appropriate using the terms putative or candidate pectate lyases.
Re: Thank you for your comments.We conducted analysis on the pectate lyase activity of PlPeL1, PlPeL1-like and mutants (new Fig. 1e).And LcPIP1 did not suppress the enzymatic activity of PlPeL1 or PlPeL1-like (new Supplementary Fig. 8c).The detailed experiments associated with pectate lyase activity have been added to the revised manuscript.
Overall, the authors use lesion diameter to account for pathogen infection, but lesions are not always circular, in particular for experiments on litchi.Using lesion area would be more accurate.
Re: Thanks for the suggestion.In revised manuscript, we conducted lesion area measurements using the ImageJ software for cases where lesions were not circular, ensuring a more precise assessment of pathogen infection.However, for circular lesions, we still used diameter measurements for statistical analysis.This proximity in the promoters for both genes, even if they are in opposite strands, could lead to a competition for expression in the WT, and thus to an increase of the expression of one of the genes when the other is knocked down.
Re: Thanks for pointing this out.We have made adjustments to the manuscript.Firstly, we have repositioned the figure that was previously mentioned as "Fig.1c" as a supplementary figure (now identified as Supplementary Fig. 4).Additionally, as per your suggestion, we have revised the manuscript by incorporating the relevant content into the discussion section (Lines 382-389).
P7, l 130-131 and Suppl Fig 5 .In order to highlight the conservation of the Asp residues, it could be more interesting to present the alignment of PlPeLs with other PeLs from other species.Related to this, the sentence in l 130 needs to be developed.
Re: Thank you for your suggestions.We have added the alignment of homologous proteins of PlPeL1 and PlPeL1-like in 15 oomycete species (no homologous protein of PlPeL1/PlPeL1-like was identified in fungi or plants).The findings from this alignment reveal that the seven D residues are also highly conserved in 24 homologous proteins of PlPeL1 and PlPeL1-like (new Supplementary Fig. 5b).We have incorporated these relevant results into the revised manuscript (Lines 123-126).
Define positive and negative controls in Figure 2a Re: Thanks!BD-53+AD-T and BD-Lam+AD-T were employed as the positive and negative controls, respectively.Detailed explanations of these controls can be found in the revised Methods (Lines 615-616) and the figure legend (Fig. 2a).
P11, l 208.The conserved motif VDMASG could be extended to VDMASGK/RYLW Re: Thank you for your suggestions.Previously, we contemplated extending the conserved motif to include VDMASGK/RYLW.However, our experiments with the deletion mutant M1 (1-75 aa) showed that it induced cell death in N. benthamiana to a similar extent as the full-length protein.This finding suggested that K/RYLW may not be the critical domain responsible for inducing plant resistance.While the conserved role of VDMASGK/RYLW motif in other functions remains unexplored, we have decided to use the VDMASG motif as it appears to be more suitable for our current study.
P11, l 209.It is not clear to me what is the contribution to the manuscript of the phylogenetic tree presented in Figure 5a.
Re: Thanks for the comments.We have expanded the scope of the phylogenetic tree from an initial 12 plant species to now encompass 24 species, including additional members from the Sapindaceae family.The phylogenetic tree and the sequence alignment together illustrate the sequence and evolutionary relationships of LcPIP1 and its homologs proteins.Importantly, we found that 11 homologous proteins of LcPIP1, spanning different groups, showed cell death-inducing activity (new Fig. 4e), suggesting that conserved role of PIP1s in inducing cell death in N. benthamiana.We have revised the related sentence for clearer expression (Lines 237-243).
P11, l 209.Provide accession numbers for all proteins in Suppl Figure 9.
Re: Thank you for your question.Firstly, we need to clarify that in our study, we did not find evidence of PlPeL1 or PlPeL1-like suppressing plant defense signaling.In this study, we observed that their expression in N. benthamiana enhanced susceptibility to Ph. capsici, consistent with their role as virulence factors in Peronophythora litchii.The pectate lyase enzyme activity of PlPeL1 and PlPeL1-like likely facilitates pathogen invasion by degrading plant cell walls, and the PlPeL1/PlPeL1-like enzymatic activity mutants lost their virulence (new Figure 1e-g).These results are consistent with previous reports of pathogen-secreted pectate lyases (Hugouvieux-Cotte-Pattat et al., 2014;Cnossen-Fassoni et al., 2013;Cho et al., 2015).
In our experiments, INF1-mediated cell death was remarkably delayed in NbPIP1-silenced N. benthamiana plants (Figure 8a).Furthermore, as a positive immune regulatory factor, LcPIP1 triggered immune activation in N. benthamiana.In our recently conducted experiments, we discovered that co-expressing PlPeL1 M1 /PlPeL1-like M1 with LcPIP1 resulted in increased electrolyte leakage in the injection area compared to the control, suggesting that PlPeL1 M1 /PlPeL1-like M1 could enhance LcPIP1-induced plant cell death (new Figure 3j).
Therefore, we believe that the response of PIP1 to both INF1 and PlPeL1/PlPeL1-like is an integral part of the plant immune response, aimed at countering pathogen invasion and activating defense pathways to restrict pathogen proliferation.
However, we have chosen to remove this result from our analysis.This decision was based on the observation of a relatively low increase induced by PlPeL1 and PlPeL1-like on NbPIP1 (1.2 and 1.39-fold, respectively), which, while statistically significant, may not provide substantial insights or contribute significantly to our overall findings.J. Microbiol. 51, 461-470 (2013).Cho, Y. et al.A pectate lyase-coding gene abundantly expressed during early stages of infection is required for full virulence in Alternaria brassicicola.Plos One. 10, e127140 (2015).
As a non-native English speaker, I understand how frustrating this can be, but the manuscript would benefit from proof-reading by a native English speaker.
Re: Thanks for the suggestion.Based on your comment, a native English speaker who is a professor of plant pathology has looked at the manuscript for language issues, which is also mentioned in the Acknowledgements section.
Reviewer #2 (Remarks to the Author): This MS describes the identification and characterisation of two secreted proteins (PlPel1 and PlLel-like proteins) from Peronophythora litchii and their target.
In short, the authors show that: 1-deletion of both genes in P. litchii results in a loss of virulence 2-Said Pl proteins interact with a small secreted protein (Pip1) in plants 3-LcPIP mediates cell death (orchestrated by a small and highly conserved motif) 4-Plant cell death resembles a PTI-type response that requires SERK3 and activate expression of known PTI responsive genes Based on these observations, the authors conclude that LcPIP is a cell death inducing peptide that interacts with two Pl effectors.
Overall, the work is presented nicely and the MS is of good written quality.Major comments are listed below: Re: We thank the reviewer for the positive comments on our data and constructive suggestions, and time for reviewing our manuscript.
-It is not clear to me what the main message of the work is and how it is important.LcPIP induces cell death when over-expressed in plants and its silencing compromises immunity to virulent pathogens.This is an interesting observation (bolstered by interesting interaction data).Does this reduced immunity phenotype extend to non-pathogenic microbes?Reduction of PTI gene expression hints at a more general impairment, but this is not explored.That's where I think exploration would lead to exciting discoveries.
Re: Thank you for your comments and suggestions.1) Our research sheds light on a novel and pivotal aspect of plant-pathogen interactions by elucidating the interaction between the pectate lyase of the plant pathogen and the novel positive immune regulator, LcPIP1.This interaction operates within a BAK1/SERK3-dependent framework.Then, we found that loss of PIP could significantly impair plant immunity in Arabidopsis, and N. benthamiana.Our result suggested that PIP is critical and general in plant immune system.Further study might reveal PIP1 as a new layer/key regulate of complexity in the plant immune response, particularly in the PTI pathways.
In revised manuscript, we have added new results to support the function of PIP1 (new Fig. 3g-i 2) In our experiments, we conducted inoculation trials using a range of nonpathogenic microbes, including P. litchii, Pyricularia angulate, Colletotrichum gloeosporioides, and Lasiodiplodia theobromae on AtPIP1 knockout Arabidopsis plants and NbPIP1-silenced N. benthamiana plants.However, the results showed that these non-pathogenic microbes failed to infect either Arabidopsis or N. benthamiana.Based on these materials, we have not yet found that PIP1-mediated immunity is related to non-pathogenic microbes.
Nevertheless, your insight has inspired us to begin to screen a broader range of non-pathogenic microbes or mutants to assess the potential susceptibility of PIP1 mutants.But it will take a long time to reveal the underlying mechanism, and we hope to reveal those findings in a future publication.
-The authors show that two Pl effectors bind Litchi (and Nb) PIP AND that disabling both effectors affect virulence.The significance of these observation is not clear.What is the consequence of PIP1 binding?Disabling of PIP1 function or PAMP perception?The authors will know that knocking out two effector coding genes could cause a virulence phenotype that has little to do with LcPIP1 signalling.Infection assays that feature knockout strains and PIP1 mutants would help address that.
Re: Thanks for the comments and suggestions.1) As reviewer mentioned, PlPeL1/PlPeL1-like possess pectate lyase activity, allowing them to degrade the plant cell wall and enhance virulence during pathogen infection.Regarding the proposal for infection assays involving knockout strains and PIP1 mutants, we understand the significance of this approach.However, its execution poses several challenges: firstly, P. litchii does not infect N. benthamiana or Arabidopsis; second, homologous proteins of PlPeL1 and PlPeL1-like are absent in Ph. capsici; third, current genetic transformation methods for Ph.infestans and litchi are difficult, which hinders the creation of knockout strains and mutants.These challenges collectively hinder our ability to perform the suggested experiments.
2) We have conducted additional experiments to further investigate the relationship between PlPeL1/PlPeL1-like and LcPIP1.The results suggested that LcPIP1 could be indirectly regulated by PlPeL1/PlPeL1-like in litchi (new Fig. 4b); PlPeL1 M1 /PlPeL1like M1 could enhance LcPIP1-induced cell death (new Fig. 3j); and PlPeL1/PlPeL1like did not disrupt the interaction between LcPIP1 and LcSERK3 (new Fig. 7f,g).In addition, silencing of NbPIP1 resulted in enhanced susceptibility of plants induced by PlPeL1 (new Fig. 3k,l).These results support the hypothesis that the interaction between PlPeL1/PlPeL1-like and PIP1s can enhance plant immunity, which is mediated by PIP1s in N. benthamiana.
It is possible that PIP1-mediated immunity is actively suppressed during infection and if so, understanding the mechanisms by which Pl disrupts its signalling would be of great interest.As it stands, however, there is not enough data to support a credible model that informs on the action of PIP1 and (possibly) Pl virulence factors.The proposed model reflect these knowledge gaps as it is fairly vague and throws up many questions.
Re: Thanks!We acknowledge the possibility that potential effectors or virulence mechanisms could actively suppress PIP1-mediated immunity during infection.In addition, it's notable that PlPeL1/PlPeL1-like also possess pectate lyase activity, allowing them to degrade the plant cell wall and enhance virulence during pathogen infection.This parallels the well-established importance of numerous cell walldegrading enzymes in facilitating pathogen invasion (Dora et al., 2022;Kubicek et al., 2014).It is important to emphasize that the intricate interactions between plants and pathogens are subject to a multitude of influences.Consequently, the mere expression of LcPIP1/NbPIP1 may not be sufficient to fully counteract the virulence potential of PlPeL1/PlPeL1-like in the natural condition.
We have new data to support virulence function of PlPeL1-mediated enzymatic activity.It is interesting how PIP1-mediated immunity is suppressed by pathogen; however, we did not find that PlPeL1/PlPeL1-like could suppress the function of PIP1.Reviewer #3 (Remarks to the Author):

References
This manuscript describes the involvement of pectate lyases (PlPeL1 and PlPeL1-like) from Peronopthora litchii in pathogenicity and a novel plant cell death-inducing protein, LcPIP1, from litchi.First, the authors created single or double deletion mutants of PlPeL genes and demonstrated that the double deletion mutant impaired the pathogenicity of P. litchi.Next, the authors investigated the interaction of PlPeLs and PIP1s.They demonstrated that the signal peptides and the VDMASG motif of LcPIP1 were essential to induce PCD in Nicotiana benthamiana.Moreover, they showed that the plant immune response by LcPIP/NbPIP was triggered via SERK3.The data presented in this manuscript is very interesting and the methods used are reasonable.The manuscript, however, has several critical issues.I would recommend this manuscript for publication in Nature Communications, provided the authors reasonably address the following points.
Re: We thank the reviewer for the positive comments on our data and constructive suggestions, and time for reviewing our manuscript.
Re: We apologize for the mistake and thank the reviewer for bringing it to our attention.The ''identity 99.78%'' reported in the initial manuscript was based on the preliminary comparison result obtained from the NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi).However, upon careful reevaluation based on full length, we have now corrected the identity to ''94%'' (Line 86).
These two pel genes can be named as PlPeL1 and PlPeL2 because the C-terminal regions of the two proteins are quite different.That makes easier to distinguish them ^c i]Z bVcjhXg^ei, O]Z Vji]dgh Ydcti ]VkZ id X]Vc\Z i]Z cVbZh Wji D higdc\an recommend.
Re: Thank you for your suggestions.However, through Illumnia sequencing, we identified 18 PeL encoding genes in P. litchii genome, based on bioinformatic analysis (Ye et al., 2016).Eighteen PeL genes were named based on the genome published by Ye et al., 2016. Subsequently, in 2020, our laboratory reassembled the P. litchii genome using both second and third-generation sequencing technologies.During this process, we identified a protein with a sequence and transcription pattern highly similar to PlPeL1.Therefore, we designated it as PlPeL1-like.Line 125: Was the full length of PlPel1-lilke protein expressed including the unique C-terminus?

References
Re: Thanks for the question.Yes, we expressed the full length of PlPel1-lilke protein in N. benthamiana leaves using agroinfiltration, and western blot (Fig. 1b or Supp.Fig. 6a) confirmed that the PlPel1-lilke protein expressed including the unique Cterminus and was larger than PlPel1, with a difference of approximately 7 KDa.
Line 142: Which means that the increase of the susceptibility was responsible for only the degradation of pectic substances (pectate)?
Re: Thank you for your question.According to previous reports, it's well-established that most plant pathogens secrete a variety of cell wall-degrading enzymes (CWDEs) to break down plant cell walls and obtain nutrients for their own growth and infection (Kikot et al., 2009).In this study, we also found that the enzymatic activity of PlPeL1/PlPeL1-like were also responsible for their virulence function (new Fig. 1e-g).However, we did not state that the susceptibility was only induced by the degradation of plant cell wall.M1 , respectively.The decision to make this change was driven by our observation of lower protein expression levels in mutants lacking the signal peptide (SP), which raised concerns about potential loss-of-function effects caused by reduced protein expression.
Regarding your question about signal peptides, both PlPeL1 M1 and PlPeL1-like M1 , as described in the revised manuscript, do contain signal peptides.We have provided this additional explanation in Line 128.
Line 166: Have you conducted the expression assay of LcPIP1 using litchi inoculated with a single or double deletion mutants of Pel genes?Apparently, Pels up-regulate NbPIP1 in N. benthamiana from the results of Supplementary Fig. 11.Whether the expression of LcPIP1 is also regulated by Pels in litchi is intriguing.
Re: Thanks for the suggestions.Recently, we investigated the expression of LcPIP1 in single or double deletion mutants-infected litchi (new Fig. 4b).The transcription level of LcPIP1 displayed less upregulation in litchi infected with double knockouts at 6 and 12 hpi, as opposed to the transcription level observed upon inoculation with the wild-type strain.
However, we noticed that LcPIP1 was up-regulated during pathogen infection.So the transcription levels of LcPIP1 decreased during infection by double-deletion mutants, which may be caused by the weakened pathogenicity of the deletion mutants.Therefore, we have added "may be" to the conclusion that LcPIP1 is regulated by PlPeL1 and PlPeL1-like in litchi, ''These results indicate that LcPIP1 expression might be indirectly regulated by PlPeL1 and PlPeL1-like in litchi.'' .This modification allows us to include or not exclude the possibility that other factors, such as the influence of pathogenicity on LcPIP1 expression, might contribute to the observed results.
Line 233: Single (PIP) and co-expressing (PIP/PeL) leaves showed the same lesion size, which raises the following question: PlPeL1 and -like have no effect on the infection of P. cspsici?I just wonder how the complex of LcPIP1/PlPeL1 is involved in pathogenicity and plant resistance.I understand that LcPIP1 indeed induced PCD in N. benthamiana from your data.Do you expect that PlPeLs from P. litchii have function to inhibit LcPIP1 from binding to SERK3 through interacting with each other in addition to degradation of pectic substances? or LcPIP is secreted to inhibit the enzymatic activity of PlPeLs in addition to the induction of immune systems?I understand that they have not completely proven yet in you work, but I believe it is necessary to describe your prediction at least.From the title of your paper, readers will imagine the effect given by the interaction between Pel and PIP toward pathogenicity and the plant immune systems.
Re: Thank you for your comments and suggestions.Our study revealed that the expression of PlPeL1/PlPeL1-like promote the infection of Ph. capsici and the knockout of these two genes led to reduced virulence in P. litchii.These results indicated that PlPel1 and PlPel1-like play critical roles in the virulence of P. litchii.However, when PlPeL1 or PlPeL1-like was co-expressed with LcPIP1 in N. benthamiana, LcPIP1-mediated plant defense compromised the virulence function of PlPeL1/PlPeL1-like.Silencing of NbPIP1 resulted in enhanced susceptibility of N. benthamiana induced by PlPeL1 (new Fig. 3k,l), and PlPeL1 M1 /PlPeL1-like M1 could enhance LcPIP1-induced cell death (new Fig. 3j).
In other word, our results unveiled the dual roles of PlPeL1/PlPeL1-like in plantpathogen interactions: enhancing pathogen virulence through pectate lyase activity while also being targeted by LcPIP1, enhancing plant immunity.It is well known that some cell wall-degrading enzymes (CWDEs) secreted by pathogens can act as both virulence factors and inducers of plant immunity.For example, GH12 protein PsXEG1, xylanase BcXyl1, pectate lyase VdPEL1 and FsPL are known to be important virulence factors while also activating the plant PTI response (Ma et al., 2017;Yang et al., 2018;Wang et al., 2023;Yang et al., 2018).
Furthermore, according to several suggestions of the reviewer, our new results showed that LcPIP1 did not interfere with PlPeL1 or PlPeL1-like enzyme activity (new Supplementary Fig. 8c).In addition, PlPeL1/PlPeL1-like did not disrupt LcPIP1 from binding to LcSERK3 through interacting with each other (new Fig. 7f,g).However, the impact of PlPeL1/PlPeL1-like on the interaction between LcPIP1 and potential receptor-like kinases (RLKs) remains unknown, and further investigation is needed in this regard.
Regarding the title, to our knowledge, this is the first report on the interaction between pectate lyases of a plant pathogen and a plant positive immune regulator.Thus, we highlight the novel interaction between pectate lyases and the positive immune regulator PIP1.
PlPeL1-like, expressing PIP1 as well, showed the same lesion size (Figure 1d-g and Figure 6ab).
Re: Thanks for the question.1) In our study, when PlPeL1 or PlPeL1-like was co-expressed with LcPIP1, the N. benthamiana leaves showed the same lesion size (new Fig. 3a,b).However, when PlPeL1 was co-expressed with LcPIP1, the relative Ph. capsici biomass was significantly smaller compared to co-expressing PlPeL1-like and LcPIP1, as determined by a Student's t-test (new Fig. 3c).Quantification of pathogen biomass provides a more detailed assessment of Ph. capsici growth and proliferation during infection, suggesting that LcPIP1 might exert a more pronounced impact on attenuating the virulence function of PlPeL1 compared to PlPeL1-like.
2) We adjusted the OD600 of the injected Agrobacterium to 0.05, 0.005, and 0.0005.Surprisingly, under lower OD600 conditions (OD600 = 0.005), lesions on leaves expressing PlPeL1 were significantly smaller than those on leaves expressing RFP (Author Response Figure 4a).Despite this, PlPeL1-like still promoted pathogen invasion more effectively than RFP.Furthermore, in OD600 = 0.0005 conditions, lesions on leaves expressing PlPeL1 and PlPeL1-like were significantly smaller than those on leaves expressing RFP (Author Response Figure 4b).At low concentrations of both PlPeL1 and PlPeL1-like, they may play a major role in inducing plant resistance.This suggests that PlPeL1-like can exert its virulence effect at lower protein levels, possibly making the plant resistance response induced by NbPIP1 less susceptible to interference by PlPeL1-like.
3) PlPeL1 showed a greater than 20-fold increase in relative expression compared to PlPeL1-like at 24 hpi (new Supplementary Fig. 1e), suggesting that the plant may require stronger suppression of PlPeL1 virulence.Based on above findings, we hypothesize that LcPIP1 may more strongly inhibit the virulence function of PlPeL1.This could explain why silencing NbPIP1 did not significantly increase the virulence of PlPeL1-like.4) Additionally, in NbPIP1-silenced plants, specific plant resistance-related genes may be upregulated and specifically recognize PlPeL1-like.However, these underlying mechanisms remain enigmatic and warrant further investigation.In the revised manuscript, we added additional discussion regarding this concern (Lines 421-429).
Line 365: As you mentioned, NbPIP1 may interact with INF1 to trigger the immune systems.Again, how about the interaction (binding) between PeL1 and PIP1?What does this interaction cause?Just to avoid the degradation of cell walls?Re: Thanks for the questions.In our experiments, INF1-mediated cell death was remarkably delayed in NbPIP1-silenced N. benthamiana plants (Fig. 8a).Furthermore, as a positive immune regulatory factor, LcPIP1 triggers cell death accompanied by reactive oxygen species (ROS) accumulation, callose deposition, and induction of defense genes in N. benthamiana.LcPIP1 interacted and perceived with PlPeL1 and PlPeL1-like to increase host resistance (new Fig. 3).Therefore, our results showed that the response of PIP1 to both INF1 and PlPeL1/PlPeL1-like will activate plant defense signaling to restrict pathogen proliferation.
As mentioned above, we conducted additional experiments.The results indicate that LcPIP1 did not affect the pectate lyase enzyme activity of PlPeL1 and PlPeL1like.Therefore, currently, there is no evidence to support the inhibition of PlPeL1/PlPeL1-like-mediated cell wall degradation by LcPIP1.
Line 379: You mentioned that NbPIP1 attenuated the susceptibility induced by PlPeL1 in line 251 (Figure 6g,h).But here, you describe that NbPIP1-silencing did not affect the susceptibility quoting Figure 6g,h.It is confusing.
Re: Thank you for your comments and questions. 1) In our revised manuscript, we conducted experiments involving the knockout of the homologous gene AtPIP1 in Arabidopsis.These atpip1 knockout mutants showed an increased susceptibility to Ph. capsici infection (new Fig. 3g-i).
2) The silencing of gene using VIGS results in a reduction in its expression, rather than complete elimination.As shown in Figure 3d, the expression level of NbPIP1 remained at 15.5% of the control (TRV-GUS).Consequently, the plant retained a certain level of recognition and defense capabilities, albeit at a diminished level.
3) Furthermore, it's crucial to consider that PlPeL1 and PlPeL1-like lack a homologous protein in Ph. capsici (new Supplementary Fig. 5b).PlPeL1 directly interacted with NbPIP1 (Fig. 2b,c), possibly enhancing the virulence function of PlPeL1 in NbPIP1 silencing plants.4) In light of the complex network of plant defense responses, it is conceivable that the effects of NbPIP1 silencing may be partially countered by compensatory mechanisms involving other proteins within the intricate defense signaling network.This intricate web of interactions and compensatory responses could collectively contribute to the observed absence of a significant difference in pathogen resistance between the NbPIP1-silenced plants and the control.5) In the revised manuscript, we added additional discussion regarding this concern (Lines 414-421).
Line 386: Can you say PlPeLs trigger the immune systems?If so, leaves expressing PlPeLs should show resistance against P. capsici somehow.Your sentence from lines 386 to 388 sounds like it is happening in litchi.I suggest that you describe the roles of PeLs from the pathogen end and those of PIP1 from the plant end, and on top of that, what the interaction of PeLs and PIP1 brings about.Please distinguish clearly whether you are describing about N. benthamiana or litchi.
Re: Thanks for the questions and suggestions.1) Our results demonstrate that PlPeL1 M1 /PlPeL1-like M1 could enhance LcPIP1induced cell death (new Fig. 3j).Furthermore, LcPIP1 expression may be modulated by PlPeL1/PlPeL1-like in litchi.These results support that the interaction between PlPeL1/PlPeL1-like and PIP1s can enhance plant immunity, which is mediated by PIP1s.However, it's important to note that PlPeL1/PlPeL1-like also possess pectate lyase activity (new Fig. 1e), allowing them to degrade the plant cell walls and enhance virulence during pathogen infection.This parallels the well-established importance of numerous cell wall-degrading enzymes in facilitating pathogen invasion (Dora et al., 2022;Kubicek et al., 2014).
Variations in the expression levels of PlPeL1 and PlPeL1-like lead to diverse impacts on plant resistance against Ph.capsici (Author Response Figure 4).Specifically, when PlPeL1/PlPeL1-like accumulation was at a lower level, plant resistance took precedence, while higher accumulation shifted the balance toward a dominance of virulence.
It is important to emphasize that the intricate interactions between plants and pathogens are subject to a multitude of influences.Consequently, the mere expression of LcPIP1/NbPIP1 may not consistently suffice to fully counteract the virulence potential of PlPeL1/PlPeL1-like under natural conditions.Pathogens can employ a diverse array of strategies, including the secretion of other effectors, to subvert or manipulate host resistance mechanisms.
This study presents a novel perspective on plant-pathogen interactions, specifically highlighting the interaction between novel plant's immune regulator, PIP1 and pathogen-secreted PeLs (PlPeL1/PlPeL1-like).We acknowledge that the mechanistic intricacies behind this interaction warrant further exploration.In ongoing research, we are using Co-IP and LC-MS/MS analyses to identify downstream resistance-associated proteins directly engaged by the interaction between PlPeL1/PlPeL1-like and LcPIP1.Particular attention is being given to proteins potentially associated with salicylic acid (SA), aiming to shed light on the potential relationships between LcPIP1 and systemic acquired resistance (SAR).2( <XXdgY^c\ id i]Z gZk^ZlZgth hj\\Zhtion, we have made efforts to provide a more explicit conclusion regarding the role of the PlPeL1/PlPeL1-like-PIP1 module in the plant-pathogen interaction (Lines 476-485).
3) We have made a concerted effort to maintain a clear distinction between the descriptions of N. benthamiana and litchi throughout the entirety of our manuscript.
, the values for CK (Fig 3a) and RFP (Fig 4) appear to be the same at all time points.This is surprising, particularly for Fig 4, because agroinfiltration should give some level of induction of PTI-related genes.

Figure 2 (
For review only) Immunoblot analysis of LcPIP1, PR1 SP -LcPIP1 {NK and LcPIP1 & SP expressed in N. benthamiana leaves.Total proteins were extracted from N. benthamiana leaves at 36 hpa.Red asterisks indicated protein bands of the correct size.Ponceau S staining of Rubisco was used to indicate loading quantity of protein samples.Author Response Figure 3 (For review only) LcPIP1 could induce cell death in N. benthamiana at low protein levels.(a) qRT-PCR analyzed the transcription levels of the C-terminal region of LcPIP1 in leaves expressing LcPIP1 or LcPIP1 {NK .Statistical analysis was conducted using the NijYZcith t-test method.(b) Total proteins were extracted from N. benthamiana leaves at 36 hpa.Red asterisks indicated protein bands of the correct size.Ponceau S staining of Rubisco was used to indicate loading quantity of protein samples.(c) N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens strains carrying HA-tagged LcPIP1 or LcPIP1 {NK constructs.Photographs were taken at 4 dpa.References: Pi, L. et al.A GÏtype lectin receptorÏlike kinase regulates the perception of oomycete apoplastic expansinÏlike proteins.J. Integr.Plant Biol.64, 183-201 (2022).Wei, W. et al.A fungal extracellular effector inactivates plant polygalacturonaseinhibiting protein.Nat. Commun.13, (2022).Xu, Y. et al.Phytophthora sojae apoplastic effector AEP1 mediates sugar uptake by mutarotation of extracellular aldose and is recognized as a MAMP.Plant Physiol.
, new Fig 4e, and new Fig 6) and tried our best to clearly describe our conclusion (Lines 476-485).
If so, leaves expressing PlPeLs should show resistance against P. capsici somehow.Your sentence from lines 386 to 388 sounds like it is happening in litchi.I suggest that you describe the roles of PeLs from the pathogen end and those of PIP1 from the plant end, and on top of that, what the interaction of PeLs and PIP1 brings about.Please distinguish clearly whether you are describing about N. benthamiana or litchi.
This is consistent with established method for lesion size quantification in Phytophthora infection studies, as documented in relevant literature(Li et al., 2023; Qiu et al., 2023; Yang et  al., 2023; Yang et al., 2022).
P7, l 118-122.The claim that up-regulation of either PlPeL1 and PlPeL1-like upon knocking out the other supports functional redundancy should be played down.In Suppl Fig 1 the authors show that both genes are separated by approx.1100 bp.