Periodic Reporting for period 1 - IMMUNE (Investigation into the Molecular Mechanisms Underlying NEmatode recognition by the Ma resistance protein in perennial plum roots)
Période du rapport: 2019-11-15 au 2021-11-14
Plant-parasitic nematodes are major root pathogens that affect drastically plant development and growth. Predominant species, such as the root-knot nematodes (RKNs) Meloidogyne spp., represent a global threat for annual and perennial crops causing huge crop losses worldwide. In Europe, RKNs were mainly controlled by toxic pesticides that are getting banned. One of the most promising alternative to pesticides is to rely on biological control and on the plant innate immunity for disease resistance in crops. The plant immune system is a multi-layered network that enables the detection of pathogens using different classes of receptors. The presence/absence of such receptor can determine whether or not the plant will be resistant/susceptible to a particular disease. At INRAE Sophia Agrobiotech Institute, an immune receptor gene Ma, from the TIR-NB-LRR (TNL) family, has been cloned from the plum tree Prunus cerasifera where it provides a broad resistance against numerous RKN species. A unique feature of Ma resides in five repeated exons encoding a large C-terminal extention. Interestingly, each of those exons carries a recently characterized post-LRR (PL) domain which is found in a single copy in many other TNLs from different plant species. The PL domain’s function remains unknown but its conservation suggests an important role in TNLs. The peculiar architecture of Ma provides a great opportunity to decipher the involvement of the PL domain in TNL–mediated immunity. Combining complementary approaches, the proposed IMMUNE project provided a first description of how Ma triggers immunity in response to RKNs in plant roots and how the PL domain participates in the recognition and signaling.
We successfully assembled the Ma wild type resistant (R) and susceptible (S) alleles from plum accession P.2175 (where Ma originates from) as well as chimeric and truncated Ma alleles using the Golden Gate cloning methods. All Ma constructs were used and transferred into the RKN susceptible plum accession P.2032 producing transgenic hairy roots expressing Ma alleles. The data obtained enabled us to conclude that the regulatory regions of the Ma S are functional and that the absence of resistance to RKN conferred by this allele lies within its coding region.
I used two other TNLs, RPS4 and N, for the PL domain analysis. I generated a selection of alleles mutated within the PL domain and identified conserved amino acids shared between those two that are required for the receptor functionality. We produced a variety of PL domain swaps between RPS4 and its paralog RPS4B. Among those, we obtained auto-active as well as non-functional alleles. Altogether those actions enabled me to conclude that the integrity of the PL domain is required for TNL receptors’ function. More precisely, the PL domain is involved in TNL receptors stabilisation in resting state, TNL activation and TNL–mediated immune signaling in plants.
We successfully assembled the Ma wild type resistant (R) and susceptible (S) alleles from plum accession P.2175 (where Ma originates from) as well as chimeric and truncated Ma alleles using the Golden Gate cloning methods. All Ma constructs were used and transferred into the RKN susceptible plum accession P.2032 producing transgenic hairy roots expressing Ma alleles. The data obtained enabled us to conclude that the regulatory regions of the Ma S are functional and that the absence of resistance to RKN conferred by this allele lies within its coding region.
I used two other TNLs, RPS4 and N, for the PL domain analysis. I generated a selection of alleles mutated within the PL domain and identified conserved amino acids shared between those two that are required for the receptor functionality. We produced a variety of PL domain swaps between RPS4 and its paralog RPS4B. Among those, we obtained auto-active as well as non-functional alleles. Altogether those actions enabled me to conclude that the integrity of the PL domain is required for TNL receptors’ function. More precisely, the PL domain is involved in TNL receptors stabilisation in resting state, TNL activation and TNL–mediated immune signaling in plants.
We have designed modules that split the Ma gene in four parts (promoter region, TIR-NB-LRR, five PL domains, and terminator region). We obtained each module of the Ma R (functional for RKN resistance) and Ma S (non-functional) alleles from P.2175. The reconstituted Ma R (Ma RRRR) and Ma S (Ma SSSS) alleles were transferred into the susceptible accession P.2032. As expected Ma RRRR provided resistance to M. incognita but Ma SSSS did not. We then swapped the promoter and terminator from Ma R and Ma S (Ma RSSR and Ma SRRS). We could detect the expression of both transcripts but only resistance to RKN in P.2032 carrying Ma SRRS. This suggests that the absence of resistance to RKN in Ma S is likely due to mutation(s) and/or deletion(s) within its coding sequence. This has to be investigated further. We generated a Ma RSRR allele which did not provide resistance RKN suggesting that such deleterious modification could lie within the TIR-NB-LRR encoding sequence. We developed an optimized protocol for hairy roots transformation with large genetic construction generated using the Golden Gate method. This protocol has been share through INRAE and will be exploited in scientific projects for the production of transgenic roots. The acquisition of such result can benefit to crops breeders and should enable to analyse many Ma alleles and Ma orthologs for resistance to RKN and transfer in crops. In parallel, we performed a functional analysis of the PL domains of the RPS4 and N TNL receptors. When we deleted the PL domain from RPS4 and N, those two receptors were non-functional. We then mutated in RPS4 and N the most conserved amino acid within the TNL PL domains. Very interestingly, mutations abolishing RPS4–mediated immunity also did for N–mediated immunity. This pointed to us that the PL domain might be involved in an important and common molecular mechanism for TNL–mediated immunity. In addition, we swapped the PL domains of RPS4 and RPS4B (the paralog of RPS4 in Arabidopsis). RPS4B carrying the PL domain of RPS4 was non-functional. Oppositely, RPS4 carrying RPS4B PL domain had an increased auto-activity. Altogether our results again support that the PL domain integrity is required for a TNL receptor to function properly. Still using RPS4 and N, we established that the PL domain not only could dimerize but could also form homomultimers. This result of great importance as it could enlarge our vision about the molecular mecanisms taking place during immune processes in plants. All those results will highly benefit to the scientifc community and be exploited for the development of futur research programmes. Indeed, interest towards the PL domain is increasing due to several very recent publications on that subject and particularly our publication available as a free access on the online plateform of the Molecular Plant-Microbe Interaction journal. I also performed an oral presentation at the 1st MoDIP congress in October 2021 where a large scientifc and professional community was present and heard about the results I obtained on Ma and the PL domain during my MSC fellowship.
We managed to understand that variations within the regulatory region of the Ma gene is not an obstacle to obtain Ma expression. We performed deletion and mutation experiments which extended our vision about the requirement of the PL domain for TNL to function. In addition, we found out that NLR domains (and particulalry the PL domain) are very specific of their own receptor for function. Based on our results, using chimeric and mutated NLR receptors involving the PL domain for crops engineering seems hardly feasible. Instead, using natural resistance against plant pathogens for breeding programmes is likely the best approach. To extend our vision, it remains to demonstrate if the PL domain is involved in intermolecular interactions and TNLs/resistosome multimersation. We paved the way to decipher whether the PL repetition provides an additive effect compared to a single PL domain. We progressed in the understanding of the Ma receptor which impacts directly the safety of Prunus crops for durable food production such as almond, peach and plum. Indeed, our results can be used for the transfer of functional Ma gene in crops for RKN natural resistance. This will facilitate eco-friendly agricultural practices, without the use of toxic pesticides, which ultimately will have a positive impact onto consumers health.