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Helper NLRs Nrc2 and Nrc3 act co-dependently with Prf/Pto and activate MAPK signaling to induce immunity in tomato

Daniel Lüdke^1*, Jiorgos Kourelis^1*, Mauricio P. Contreras^1*, Yu Sugihara^1*, Jose Salguero-Linares^1*, AmirAli Toghani^1*, Andres Posbeyikian^1*, Jogi Madhuprakash^1* and Sophien Kamoun^1*

¹The Sainsbury Laboratory, University of East Anglia, Norwich, UK

^*All authors contributed equally to the review; authors are ordered randomly, except for the last author's position

The paper by Ning Zhang and colleagues stands as a robust addition to the NLR literature. It’s an old-style study—meticulous, rigorously controlled, and with results orthogonally replicated with independent experiments. This is a refreshing deviation from the contemporary trend of over-extended papers replete with questionable experiments that we often see in current publications.

Summary of study:

They study how the sensor NLR immune receptor Prf and its kinase partner Pto work with the helper NLRs NRC2 and NRC3 in the native host of tomato to confer resistance to Pseudomonas syringae. Although, a genetic link has previously been made between these proteins, the work has generally involved the model system Nicotiana benthamiana and mostly didn’t include the bacterial pathogen P. syringae pv. tomato (Pst).

Main findings:

1/ The study confirms the redundancy between NRC2 and NRC3 in relation to Prf/Pto. However, when both are mutated, the result is fully susceptible to the level observed with a prf mutant tomato.

2/ They identify a molecular connection between NRC2/3 and MAPK signaling activated by AvrPto/AvrPtoB. MAPK are genetically downstream of NRC2/3.

3/ NRC2 and NRC3 quantitatively contribute to resistance against certain Pst strains, even those without AvrPto and AvrPtoB, reinforcing the idea that these helper NLRs are central nodes in immune pathways. This indicates that other previously uncharacterized NRC2/3-dependent immune receptor(s) against Pst are present in tomato.

4/ An intriguing observation is made that for NRC3 to become autoactive, it requires Prf, suggesting a mutual dependency between these proteins.

Introduction:

Contrary to the statement in the introduction, NRCs aren't typically recognized as essential for the TIR-NLR (TNL) response.

NRCX isn't predominantly viewed as a helper contrary to what’s mentioned in the introduction. At present, it's more accurately described as a modulator of other NRCs (Adachi et al., 2023).

Furthermore, while Wu et al., 2017 indeed relied solely on VIGS, subsequent papers on the NRC system have utilized various CRISPR mutant lines of nrc2/3/4 in N. benthamiana for their research.

Results:

The clarity of the results in Figure 1B is commendable, but they represent only one of the three replicates. This presentation could be misleading since the other replicates are neither displayed nor discussed. This happens throughout all the bacterial growth assays carried out in the paper. We would encourage incorporating the results from all replicates in the graphs.

It is praiseworthy that they employed independent CRISPR mutants as depicted in Fig. 1/Fig. S4. Many plant studies unfortunately don't adhere to this high-quality standard. Therefore, merging Fig. S4 with Fig. 1 would enhance the display of the findings, highlighting the consistent phenotypes across different mutants.

When interpreting the experiments on NRC2 and NRC3's contribution to PRR-triggered immunity (PTI) responses, caution is necessary. Without using a hrcC mutant, any contribution of NRC2/3 to PTI might be overshadowed by PTI suppressing Type 3-effectors (T3Es) of Pst.

The heading "NRC2 and NRC3 do not contribute to typical PTI responses" should be revised for precision since only flg22 and flgII-28 were assessed, while other PAMPs exist in DC3000. A suggested modification: " NRC2 and NRC3 do not contribute to flg22 and flgII-28-induced responses."

Fig. 4B's readability is compromised by the shaded boxes. Consider opting for color coding or scatter plots using one of the many R scripts that are widely available.

In Fig. 5B, it would be beneficial to specify the time point at which the NRC3 protein was extracted, especially considering the occurrence of cell death.

For Fig. 5B-C, could NRC3 autoactive be unstable in the hairpin-silenced hpPrf line? The possibility that Prf might destabilize NRC3 autoactive would then contrast with the model presented in Fig. 5D. Western blots would help test such a possibility.

The experiments in Fig. 5C with hpPrf1 would benefit from the inclusion of a positive control for hypersensitive response (HR) using any NLR that is independent of Prf.

The section positing that "NRC3 autoactive requires Prf" may be the most tentative. Ideally, this would necessitate further exploration. However, it does provide value by genetically associating the two proteins. Notably, this was conducted with a hpPrf line and not a CRISPR/Cas9 line, and in N. benthamiana rather than tomato. This contrasts with the other orthogonally replicated experiments in the paper.

This experiment would also benefit by complementation with a silencing-resilient Prf construct.

The model in Figure 5D resembles a graphical abstract but is curiously positioned ahead of Figure 6. Why not place the model at the conclusion, encapsulating all findings, including potential unknown NLR/PRR acting via NRC2/3 independent of AvrPto/AvrPtoB?

It's worth noting that NRC3 has been linked to the HR induced by PRRs of the Cf-2/4 class (Kourelis et al., 2022). Consequently, the phenotype observed with Pst strains devoid of AvrPto and AvrPtoB might be attributed to unidentified cell surface receptors, in addition to undiscovered sensor NLRs. It might be worthwhile to include this possibility into the discussion. The quantitative nature of the phenotype may also point to a weak-acting PRR.

Competing interests

The author declares that they have no competing interests.