PREreview of Rpv2is part of a cluster of NLRs specific toVitis rotundifoliaand confers extreme resistance to grapevine downy mildew

Marsan et al. present a detailed genetic and molecular analysis of Rpv2, a resistance locus from Vitis rotundifolia cv. Trayshed, conferring robust resistance to Plasmopara viticola, the causal agent of grapevine downy mildew. Through a combination of fine-mapping, chromosome painting, and transcriptomic analyses across multiple segregating populations, the authors narrow down Rpv2 to a ~250 kb interval on chromosome 18 containing a cluster of NLR genes. Two TIR-NBS-LRR genes (TNLs), highly conserved in resistant genotypes and absent or divergent in susceptible Vitis species, are proposed as strong candidates. Notably, the resistance conferred by Rpv2 is effective early in infection and often does not result in visible necrosis, distinguishing it from other known Rpv loci. The study also clarifies the genetic separation between Rpv2 and Run2.2, which is important for pyramiding resistance genes in breeding programs.

This is a well-executed and timely study that will be of broad interest to plant pathologists and grapevine breeders. The genetic and molecular resources generated are valuable, and the data are presented clearly. The identification of a novel resistance locus with what appears to be "extreme resistance" to P. viticola is an important contribution. We commend the authors for the breadth of the mapping work and the integration of cytogenetics and transcriptomics.

Although the manuscript is of high quality, we have several suggestions that we feel could improve clarity, consistency, and interpretation, especially regarding terminology and framing. We hope the authors will find these constructive.

Major Comments

1. Clarification and justification of “extreme resistance”. The term "extreme resistance" is central to the paper, yet its definition is not explicitly provided. Traditionally, "extreme resistance" (e.g., the Rx gene in potato) refers to a form of resistance that blocks pathogen development with no visible cell death. We suggest the authors introduce this concept clearly in the Introduction and discuss whether Rpv2 meets these criteria. Moreover, the authors use Extreme and Total resistance in a seemingly interchangeable manner. It would be useful to clarify if these are indeed interchangeable or what the basis for using one term or another is.

2. Framing of NLRs and resistance durability (Introduction, lines 85–88) The Introduction currently frames NLRs as being “renowned for being overcome.” This is overly negative and risks misrepresenting the value of NLRs in breeding. Consider rephrasing to acknowledge both their effectiveness and limitations: for example, “NLRs confer robust and often race-specific resistance, yet single resistance genes may be overcome by rapidly evolving pathogens. Therefore, identification of new NLRs and strategic deployment—such as stacking—are critical for sustainable disease resistance.”

3. Consistency in terminology and domain nomenclature

   • Define key domain abbreviations (TIR, NB-ARC, TNL) at their first mention in the main text rather than in figure legends.

   • Use consistent terminology for NLRs throughout. Prefer “nucleotide-binding leucine-rich repeat receptors (NLRs)” for NLRs instead of switching between NB-LRRs, NOD-like receptors, and NBS. Also “TIR-NLRs” is preferred over TNLs to reduce the number of acronyms and make this more understandable by a broader audience.

   • Similarly, ensure consistent use of NB-ARC vs. NB domain. NB-ARC is preferred.

4. Definition of orthology in Figure 6 and related text The authors state that orthologs of the two candidate genes were not found in other Vitis species. Please clarify the basis for orthology assessment (e.g., sequence identity threshold, synteny, reciprocal best BLAST, etc.). This is particularly important as the absence of orthologs underpins the speculation of the two TIR-NLRs that are candidates for Rpv2.

5. Functional validation While the mapping and expression data are compelling, confirmation of the causal gene would greatly strengthen the conclusions. We appreciate that VIGS or transgenic validation may be technically challenging in grapevine and beyond the current scope, but a brief discussion of these limitations and future directions would be welcome.

Minor Suggestions

• Title: Consider removing “extreme resistance” from the title.

• Lines 82–86: Rephrase to “Makes it possible to understand…”

• Line 85: Clarify that these genes have not yet been identified.

• Line 112–113: “NLRs are often associated with quantitative resistance” — this is not accurate. Most NLRs confer qualitative, race-specific resistance. Please revise or provide a reference supporting this claim.

• Line 162: Rephrase to “Per leaf disc”

• Line 334: Rephrase to “We performed microscopy…”

• Line 341: Rephrase to “Quantification of the numbers of stomata…”

• Line 423–424: Rephrase to “Only two genes encoding full-length proteins”

• Line 443 and elsewhere: Ensure NB-ARC domain naming is consistent throughout.

Figures

• Figure 1: 

Define “logarithm of the odds (LOD)” and “LG” in the first instance.

• Figure 2:

Panel B appears to be a reference scale for panel C. However, clarification of what MYC0 to MYC4 represent would be helpful, as these are not clearly defined in the manuscript. Are they qualitative or quantitative infection scores?

Panel C: Y-axis label is missing. Clarify whether any statistical analyses were performed on this data.

• Figure 3: 

Instead of just “18,” label as “Linkage Group 18” for clarity.

• Figure 4: Label Y-axis codes (e.g., 1771P, 11137R) to clarify what they represent (e.g., genotypes, parental lines).

• Figure 5: Panel A: Consider adding a chromosome label directly on the schematic. Panel C: Add “RPKM” to Y-axis label.

• Figure 6: Clarify how orthologs and lack thereof were defined (e.g., based on synteny or sequence similarity?). Ensure gene model naming and terminology are consistent across panels.

Discussion

• Line 453 / 460: Clearly define “extreme” vs. “total” vs. “partial” resistance. Is the distinction based on timing, presence/absence of hypersensitive response, or quantitative vs. qualitative pathogen suppression?

• Line 475: Why are some genes referred to as “NLR-like”? If these are truncated or pseudogenes, please clarify.

• Literature context: Consider discussing other TIR-NLR or NLRs known to confer downy mildew resistance in other species, to place Rpv2 in a broader context.

We hope these suggestions help improve the clarity and impact of this valuable manuscript.

Competing interests

The authors declare that they have no competing interests.