Rice Yellow Mottle Virus resistance by genome editing of the Oryza sativa L. ssp. japonica nucleoporin gene OsCPR5.1 but not OsCPR5.2.

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (Oryza glaberrima), however, introgression into Oryza sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single and double knockout mutants showed neither substantial growth defects nor symptoms indicative lesion mimic phenotypes, possibly reflecting functional differentiation. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

Rapid evolution of an RNA virus to escape recognition by a rice nucleotide-binding and leucine-rich repeat domain immune receptor.

Viral diseases are a major limitation for crop production, and their control is crucial for sustainable food supply. We investigated by a combination of functional genetics and experimental evolution the resistance of rice to the rice yellow mottle virus (RYMV), which is among the most devastating rice pathogens in Africa, and the mechanisms underlying the extremely fast adaptation of the virus to its host. We found that the RYMV3 gene that protects rice against the virus codes for a nucleotide-binding and leucine-rich repeat domain immune receptor (NLRs) from the Mla-like clade of NLRs. RYMV3 detects the virus by forming a recognition complex with the viral coat protein (CP). The virus escapes efficiently from detection by mutations in its CP, some of which interfere with the formation of the recognition complex. This study establishes that NLRs also confer in monocotyledonous plants immunity to viruses, and reveals an unexpected functional diversity for NLRs of the Mla clade that were previously only known as fungal disease resistance proteins. In addition, it provides precise insight into the mechanisms by which viruses adapt to plant immunity and gives important knowledge for the development of sustainable resistance against viral diseases of cereals.

Allele mining unlocks the identification of RYMV resistance genes and alleles in African cultivated rice.

BACKGROUND: Rice yellow mottle virus (RYMV) is a major rice pathogen in Africa. Three resistance genes, i.e. RYMV1, RYMV2 and RYMV3, have been previously described. RYMV1 encodes the translation initiation factor eIF(iso)4G1 and the best candidate genes for RYMV2 and RYMV3 encode a homolog of an Arabidopsis nucleoporin (CPR5) and a nucleotide-binding domain and leucine-rich repeat containing domain (NLR) protein, respectively. High resistance is very uncommon in Asian cultivated rice (Oryza sativa), with only two highly resistant accessions identified so far, but it is more frequent in African cultivated rice (Oryza glaberrima). RESULTS: Here we report the findings of a resistance survey in a reference collection of 268 O. glaberrima accessions. A total of 40 resistant accessions were found, thus confirming the high frequency of resistance to RYMV in this species. We analysed the variability of resistance genes or candidate genes in this collection based on high-depth Illumina data or Sanger sequencing. Alleles previously shown to be associated with resistance were observed in 31 resistant accessions but not in any susceptible ones. Five original alleles with a frameshift or untimely stop codon in the candidate gene for RYMV2 were also identified in resistant accessions. A genetic analysis revealed that these alleles, as well as T-DNA insertions in the candidate gene, were responsible of RYMV resistance. All 40 resistant accessions were ultimately linked to a validated or candidate resistance allele at one of the three resistance genes to RYMV. CONCLUSION: This study demonstrated that the RYMV2 resistance gene is homologous to the Arabidopsis CPR5 gene and revealed five new resistance alleles at this locus. It also confirmed the close association between resistance and an amino-acid substitution in the leucine-rich repeat of the NLR candidate for RYMV3. We also provide an extensive overview of the genetic diversity of resistance to RYMV in the O. glaberrima species, while underlining the contrasted pattern of diversity between O. glaberrima and O. sativa for this trait. The different resistance genes and alleles will be instrumental in breeding varieties with sustainable field resistance to RYMV.

Fine mapping of RYMV3: a new resistance gene to Rice yellow mottle virus from Oryza glaberrima.

KEY MESSAGE: A new resistance gene against Rice yellow mottle virus was identified and mapped in a 15-kb interval. The best candidate is a CC-NBS-LRR gene. Rice yellow mottle virus (RYMV) disease is a serious constraint to the cultivation of rice in Africa and selection for resistance is considered to be the most effective management strategy. The aim of this study was to characterize the resistance of Tog5307, a highly resistant accession belonging to the African cultivated rice species (Oryza glaberrima), that has none of the previously identified resistance genes to RYMV. The specificity of Tog5307 resistance was analyzed using 18 RYMV isolates. While three of them were able to infect Tog5307 very rapidly, resistance against the others was effective despite infection events attributed to resistance-breakdown or incomplete penetrance of the resistance. Segregation of resistance in an interspecific backcross population derived from a cross between Tog5307 and the susceptible Oryza sativa variety IR64 showed that resistance is dominant and is controlled by a single gene, named RYMV3. RYMV3 was mapped in an approximately 15-kb interval in which two candidate genes, coding for a putative transmembrane protein and a CC-NBS-LRR domain-containing protein, were annotated. Sequencing revealed non-synonymous polymorphisms between Tog5307 and the O. glaberrima susceptible accession CG14 in both candidate genes. An additional resistant O. glaberrima accession, Tog5672, was found to have the Tog5307 genotype for the CC-NBS-LRR gene but not for the putative transmembrane protein gene. Analysis of the cosegregation of Tog5672 resistance with the RYMV3 locus suggests that RYMV3 is also involved in Tog5672 resistance, thereby supporting the CC-NBS-LRR gene as the best candidate for RYMV3.

An extensive analysis of the African rice genetic diversity through a global genotyping.

KEY MESSAGE: We present here the first curated collection of wild and cultivated African rice species. For that, we designed specific SNPs and were able to structure these very low diverse species. Oryza glaberrima, the cultivated African rice, is endemic from Africa. This species and its direct ancestor, O. barthii, are valuable tool for improvement of Asian rice O. sativa in terms of abiotic and biotic stress resistance. However, only a few limited studies about the genetic diversity of these species were performed. In the present paper, and for the first time at such extend, we genotyped 279 O. glaberrima, selected both for their impact in current breeding and for their geographical distribution, and 101 O. barthii, chosen based on their geographic origin, using a set of 235 SNPs specifically designed for African rice diversity. Using those data, we were able to structure the individuals from our sample in three populations for O. barthii, related to geography, and two populations in O. glaberrima; these two last populations cannot be linked however to any currently phenotyped trait. Moreover, we were also able to identify misclassification in O. glaberrima as well as in O. barthii and identified new form of O. sativa from the set of African varieties.

A recessive resistance to rice yellow mottle virus is associated with a rice homolog of the CPR5 gene, a regulator of active defense mechanisms.

RYMV2 is a major recessive resistance gene identified in cultivated African rice (Oryza glaberrima) which confers high resistance to the Rice yellow mottle virus (RYMV). We mapped RYMV2 in an approximately 30-kb interval in which four genes have been annotated. Sequencing of the candidate region in the resistant Tog7291 accession revealed a single mutation affecting a predicted gene, as compared with the RYMV-susceptible O. glaberrima CG14 reference sequence. This mutation was found to be a one-base deletion leading to a truncated and probably nonfunctional protein. It affected a gene homologous to the Arabidopsis thaliana CPR5 gene, known to be a defense mechanism regulator. Only seven O. glaberrima accessions showing this deletion were identified in a collection consisting of 417 accessions from three rice species. All seven accessions were resistant to RYMV, which is an additional argument in favor of the involvement of the deletion in resistance. In addition, fine mapping of a resistance quantitative trait locus in O. sativa advanced backcrossed lines pinpointed a 151-kb interval containing RYMV2, suggesting that allelic variants of the same gene may control both high and partial resistance.

Identification of a second major resistance gene to Rice yellow mottle virus, RYMV2, in the African cultivated rice species, O. glaberrima.

Rice yellow mottle virus (RYMV) is the most damaging rice-infecting virus in Africa. However, few sources of high resistance and only a single major resistance gene, RYMV1, are known to date. We screened a large representative collection of African cultivated rice (Oryza glaberrima) for RYMV resistance. Whereas high resistance is known to be very rare in Asian cultivated rice (Oryza sativa), we identified 29 (8%) highly resistant accessions in O. glaberrima. The MIF4G domain of RYMV1 was sequenced in these accessions. Some accessions possessed the rymv1-3 or rymv1-4 recessive resistance alleles previously described in O. glaberrima Tog5681 and Tog5672, respectively, and a new allele, rymv1-5, was identified, thereby increasing the number of resistance alleles in O. glaberrima to three. In contrast, only a single allele has been reported in O. sativa. Markers specific to the different alleles of the RYMV1 gene were developed for marker-assisted selection of resistant genotypes for disease management. In addition, the presence of the dominant susceptibility allele (Rymv1-1) in 15 resistant accessions suggests that their resistance is under different genetic control. An allelism test involving one of those accessions revealed a second major resistance gene, i.e., RYMV2. The diversity of resistance genes against RYMV in O. glaberrima species is discussed in relation to the diversification of the virus in Africa.