Development and validation of functional marker targeting an InDel in the major rice blast disease resistance gene Pi54 (Pik h )

Rice blast is one of the most devastating diseases affecting the rice crop throughout the world. In molecular breeding for host plant resistance, functional markers are very useful for enhancing the precision and accuracy in marker-assisted selection (MAS) of target gene(s) with minimum effort, time and cost. Pi54 (which was earlier known as Pik ^h ) is one of the major blast resistance genes and has been observed to show resistance against many isolates of the blast pathogen in India. The gene has been cloned through map-based strategy and encodes a nucleotide-binding site?Cleucine-rich repeat (NBS?CLRR) domain-containing protein. In the present study, we carried out allele mining for this gene and identified a 144-bp insertion/deletion (InDel) polymorphism in the exonic region of the gene. A PCR-based co-dominant molecular marker targeting this InDel, named Pi54 MAS, was developed. Pi54 MAS was observed to perfectly co-segregate with blast resistance in a mapping population with no recombinants. Validation of this marker in 105 genotypes which are either susceptible or resistant to rice blast disease showed that the marker is polymorphic in most of the resistant?Csusceptible genotype combinations and is more accurate than the earlier reported markers for Pi54. Hence this functional, co-dominant marker is suggested for routine deployment in MAS of Pi54 in breeding programs.     

Functional complementation of rice blast resistance gene Pi-k h (Pi54) conferring resistance to diverse strains of Magnaporthe oryzae

Blast disease of rice, caused by Magnaporthe oryzae is an explosive disease that can spread rapidly in conducive conditions. R-gene mediated resistance offers an environmentally sustainable solution for management of this important disease of rice. We have earlier identified a unique R-gene of rice, on chromosome 11 of Oryza sativa ssp. indica cultivar Tetep. In this study we report functional validation of the Pi-k ^h (Pi54) gene using complementation assay. The blast resistance candidate gene Pi-k ^h (Pi54) was cloned into a plant transformation vector and the construct was used to transform a japonica cultivar of rice Taipei 309, which is susceptible to M. oryzae. Transgenic lines containing Pi-k ^h (Pi54) gene were found to confer high degree of resistance to diverse isolates of M. oryzae. The callose deposition was analyzed and compared between the transgenic and non-transgenic rice plants and widespread deposition was observed at the infection sites in plants showing incompatible interaction. Successful complementation of Pi-k ^h (Pi54) gene confirmed that the gene is responsible for resistance to M. oryzae in transgenic lines developed during this study. Expression analysis of the gene in resistant plants revealed that the gene is pathogen inducible in nature and is not expressed constitutively. Detection of callose deposition in resistant plants containing Pi-k ^h (Pi54) gene implicates its involvement in the initiation of defense response cascade.     

The blast resistance gene Pi37 encodes a nucleotide binding site leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1

The resistance (R) gene Pi37, present in the rice cultivar St. No. 1, was isolated by an in silico map-based cloning procedure. The equivalent genetic region in Nipponbare contains four nucleotide binding site-leucine-rich repeat (NBS-LRR) type loci. These four candidates for Pi37 (Pi37-1, -2, -3, and -4) were amplified separately from St. No. 1 via long-range PCR, and cloned into a binary vector. Each construct was individually transformed into the highly blast susceptible cultivar Q1063. The subsequent complementation analysis revealed Pi37-3 to be the functional gene, while -1, -2, and -4 are probably pseudogenes. Pi37 encodes a 1290 peptide NBS-LRR product, and the presence of substitutions at two sites in the NBS region (V239A and I247M) is associated with the resistance phenotype. Semiquantitative expression analysis showed that in St. No. 1, Pi37 was constitutively expressed and only slightly induced by blast infection. Transient expression experiments indicated that the Pi37 product is restricted to the cytoplasm. Pi37-3 is thought to have evolved recently from -2, which in turn was derived from an ancestral -1 sequence. Pi37-4 is likely the most recently evolved member of the cluster and probably represents a duplication of -3. The four Pi37 paralogs are more closely related to maize rp1 than to any of the currently isolated rice blast R genes Pita, Pib, Pi9, Pi2, Piz-t, and Pi36.     

The identification of Pi50(t), a new member of the rice blast resistance Pi2/Pi9 multigene family

The deployment of broad-spectrum resistance genes is the most effective and economic means of controlling blast in rice. The cultivar Er-Ba-Zhan (EBZ) is a widely used donor of blast resistance in South China, with many cultivars derived from it displaying broad-spectrum resistance against blast. Mapping in a set of recombinant inbred lines bred from the cross between EBZ and the highly blast-susceptible cultivar Liangjiangxintuanheigu (LTH) identified in EBZ a blast resistance gene on each of chromosomes 1 (Pish), 6 (Pi2/Pi9) and 12 (Pita/Pita-2). The resistance spectrum and race specificity of the allele at Pi2/Pi9 were both different from those present in other known Pi2/Pi9 carriers. Fine-scale mapping based on a large number of susceptible EBZ?×?LTH F(2) and EBZ?×?LTH BC(1)F(2) segregants placed the gene within a 53-kb segment, which includes Pi2/Pi9. Sequence comparisons of the LRR motifs of the four functional NBS-LRR genes within Pi2/Pi9 revealed that the EBZ allele is distinct from other known Pi2/Pi9 alleles. As a result, the gene has been given the designation Pi50(t).     

Identification of the blast resistance gene Pit in rice cultivars using functional markers

DNA markers that allow for identification of resistance genes in rice germplasm have a great advantage in resistance breeding because they can assess the existence of the genes without laborious inoculation tests. Functional markers (FMs), which are designed from functional polymorphisms within the sequence of genes, are unaffected by nonfunctional allelic variation and make it possible to identify an individual gene. We previously showed that the resistance function of the rice blast resistance gene Pit in a resistant cultivar, K59, was mainly acquired by up-regulated promoter activity through the insertion of a long terminal repeat (LTR) retrotransposon upstream of Pit. Here, we developed PCR-based DNA markers derived from the LTR-retrotransposon sequence and used these markers to screen worldwide accessions of rice germplasm. We identified 5 cultivars with the LTR-retrotransposon insertion out of 68 rice accessions. The sequence and expression pattern of Pit in the five cultivars were the same as those in K59 and all showed Pit-mediated blast resistance. The results suggest that the functional Pit identified using the markers was derived from a common progenitor. Additionally, comparison of the Pit coding sequences between K59 and susceptible cultivars revealed that one nucleotide polymorphism, which caused an amino acid substitution, offered another target for a FM. These results indicate that our DNA markers should enhance prediction of Pit function and be applicable to a range of rice varieties/landraces cultivated in various regions worldwide and belonging to the temperate japonica, tropical japonica, and indica groups.     

A multiple gene complex on rice chromosome 4 is involved in durable resistance to rice blast

Quantitative trait loci (QTLs) for resistance to rice blast offer a potential source of durable disease resistance in rice. However, few QTLs have been validated in progeny testing, on account of their small phenotypic effects. To understand the genetic basis for QTL-mediated resistance to blast, we dissected a resistance QTL, qBR4-2, using advanced backcross progeny derived from a chromosome segment substitution line in which a 30- to 34-Mb region of chromosome 4 from the resistant cultivar Owarihatamochi was substituted into the genetic background of the highly susceptible Aichiasahi. The analysis resolved qBR4-2 into three loci, designated qBR4-2a, qBR4-2b, and qBR4-2c. The sequences of qBR4-2a and qBR4-2b, which lie 181 kb apart from each other and measure, 113 and 32 kb, respectively, appear to encode proteins with a putative nucleotide-binding site (NBS) and leucine-rich repeats (LRRs). Sequence analysis of the donor allele of qBR4-2a, the region with the largest effect among the three, revealed sequence variations in the NBS-LRR region. The effect of qBR4-2c was smallest among the three, but its combination with the donor alleles of qBR4-2a and qBR4-2b significantly enhanced blast resistance. qBR4-2 comprises three tightly linked QTLs that control blast resistance in a complex manner, and thus gene pyramiding or haplotype selection is the recommended strategy for improving QTL-mediated resistance to blast disease through the use of this chromosomal region.     

Pi35(t), a new gene conferring partial resistance to leaf blast in the rice cultivar Hokkai 188

The japonica rice cultivar Hokkai 188 shows a high level of partial resistance to leaf blast. For mapping genes conferring the resistance, a set of 190 F₂ progeny/F₃ families was developed from the cross between the indica rice cultivar Danghang-Shali, with a low level of partial resistance, and Hokkai 188. Partial resistance to leaf blast in the F₃ families was assessed in upland nurseries. From a primary microsatellite (SSR) linkage map and QTL analysis using a subset of 126 F₂ progeny/F₃ families randomly selected from the above set, one major QTL located on chromosome 1 was detected in the vicinity of SSR marker RM1216. This QTL was responsible for 69.4% of the phenotypic variation, and Hokkai 188 contributed the resistance allele. Segregation analysis in the F₃ families for partial resistance to leaf blast was in agreement with the existence of a major gene, and the gene was designated as Pi35(t). Another QTL detected on chromosome 8 was minor, explained 13.4% of the phenotypic variation, and an allele of Danghang-Shali increased the level of resistance in this QTL. Additional SSR markers of the targeted Pi35(t) region were further surveyed in the 190 F₂ plants, and Pi35(t) was placed in a 3.5-cM interval flanked by markers RM1216 and RM1003.     

Molecular mapping of the blast resistance gene, Pi44(t), in a line derived from a durably resistant rice cultivar

 A recombinant inbred line derived from a cross between CO39 and ‘Moroberekan’, RIL276, was found to be resistant to lineage 44 isolates of Pyricularia grisea in the Philippines. One hundred F₂ individuals were obtained from a backcross of RIL276 and CO39. Phenotypic analysis showed that RIL276 carries a single locus, tentatively named Pi44(t), conferring complete resistance to lineage 44 isolates of P. grisea. RFLP probes, STS primers and AFLP markers were applied to identify DNA markers linked to Pi44(t). Neither RFLP nor STS-PCR analysis gave rise to DNA markers linked to the locus. Using bulk segregant AFLP analysis, however, two dominant AFLP markers (AF₃₄₈ and AF₃₄₉) linked to Pi44(t) were identified. AF₃₄₉ and AF₃₄₈ were located at 3.3±1.5 cM and 11±3.5 cM from Pi44(t), respectively. These markers were mapped on chromosome 11 using an F₂ population derived from a cross between ‘Labelle’ and ‘Black Gora’. The location of AF₃₄₈ on chromosome 11 was confirmed using another F₂ mapping population derived from IR40931-26-3-3-5/ PI543851. DNA products at the loci linked to Pi44(t) were amplified from RIL276, ‘Labelle’ and PI543851 using the same primer pairs used to amplify AF₃₄₉ and AF₃₄₈. Sequence analysis of these bands showed 100% identity between lines. This result indicates that these AFLP markers could be used for the comparison of maps or assignment of linkage groups to chromosomes. Received: 12 May 1998 / Accepted: 13 November 1998     

Early and specific gene expression triggered by rice resistance gene Pi33 in response to infection by ACE1 avirulent blast fungus

* Our view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction. * Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance. * One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene-for-gene interaction involving Nipponbare resistant cultivar. * Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance.     

The in silico map-based cloning of Pi36, a rice coiled-coil nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast fungus

The indica rice variety Kasalath carries Pi36, a gene that determines resistance to Chinese isolates of rice blast and that has been located to a 17-kb interval on chromosome 8. The genomic sequence of the reference japonica variety Nipponbare was used for an in silico prediction of the resistance (R) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present. The three candidate genes were amplified from the genomic DNA of a number of varieties by long-range PCR, and the resulting amplicons were inserted into pCAMBIA1300 and/or pYLTAC27 vectors to determine sequence polymorphisms correlated to the resistance phenotype and to perform transgenic complementation tests. Constructs containing each candidate gene were transformed into the blast-susceptible variety Q1063, which allowed the identification of Pi36-3 as the functional gene, with the other two candidates being probable pseudogenes. The Pi36-encoded protein is composed of 1056 amino acids, with a single substitution event (Asp to Ser) at residue 590 associated with the resistant phenotype. Pi36 is a single-copy gene in rice and is more closely related to the barley powdery mildew resistance genes Mla1 and Mla6 than to the rice blast R genes Pita, Pib, Pi9, and Piz-t. An RT-PCR analysis showed that Pi36 is constitutively expressed in Kasalath.     

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC₁F₁ generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC₁F₁ generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC₁F₃ generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC₁F₅ generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC₁F₅ generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.     

Direct interaction of resistance gene and avirulence gene products confers rice blast resistance

Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gene relationship. Pi-ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine-rich domain (LRD) at the C-terminus. AVR-Pita is predicted to encode a metalloprotease with an N-terminal secretory signal and pro-protein sequences. AVR-Pita(176) lacks the secretory and pro-protein sequences. We report here that transient expression of AVR-Pita(176) inside plant cells results in a Pi-ta-dependent resistance response. AVR-Pita(176) protein is shown to bind specifically to the LRD of the Pi-ta protein, both in the yeast two-hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi-ta LRD or in the AVR-Pita(176) protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR-Pita(176) protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta-mediated defense response.     

The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication

? The rice-rice blast pathosystem is of great interest, not only because of the damaging potential of rice blast to the rice crop, but also because both the pathogen and its host are experimentally amenable. The rice blast resistance gene Pik, which is one of the five classical alleles located at the Pik locus on the long arm of chromosome 11, confers high and stable resistance to many Chinese rice blast isolates. ? The isolation and functional characterization of Pik were performed in the present study through genetic and genomic approaches. ? A combination of Pik-1 and Pik-2 is required for the expression of Pik resistance. Both Pik-1 and Pik-2 encode coiled-coil nucleotide binding site leucine-rich repeat (NBS-LRR) proteins, and each shares a very high level of protein identity with corresponding proteins encoded by the Pik-m and Pik-p alleles. Pik could be distinguished from other Pik alleles, including Pik-m and Pik-p, by the allele-specific, single-nucleotide polymorphism T1-2944G. ? The coupled genes probably did not evolve as a result of a duplication event, and are far from any NBS-LRR R gene characterized. Pik is a younger allele at the locus that probably emerged after rice domestication.     

The wheat durable,multipathogen resistance gene Lr34 confers partial blast resistance in rice

The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain‐of‐function mutations in an ATP‐binding cassette transporter gene. An Lr34‐like fungal disease resistance with a similar broad‐spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34‐expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence‐based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad‐spectrum disease resistance against the most devastating fungal disease of rice.