High-resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea

In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ^h ) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F₂ individuals, we mapped the Pi-k ^h gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ^h gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ^h gene revealed that its expression is pathogen inducible.     

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 diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo–devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion–deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pi_non/syn was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.     

Sequence variation of avirulence gene AVR-Pita1 in rice blast fungus, Magnaporthe oryzae

The interaction between rice, Oryza sativa, and rice blast fungus, Magnaporthe oryzae, is triggered by an interaction between the protein products of the host resistant gene, and the pathogen avirulence gene. This interaction follows the ‘gene-for-gene' concept. The resistant gene has effectively protected rice plants from rice blast infection. However, the resistant genes usually break down several years after the release of the resistant rice varieties because the fungus has evolved to new races. The objective of this study is to investigate the nucleotide sequence variation of the AVR-Pita1 gene that influences the adaption of rice blast fungus to overcome the resistant gene, Pi-ta. Thirty rice blast fungus isolates were collected in 2005 and 2010 from infected rice plants in northern and northeastern Thailand. The nucleotide sequences of AVR-Pita1 were amplified and analyzed. Phylogenetic analysis was conducted using the MEGA 5.0 program. The results showed a high level of nucleotide sequence polymorphisms and the positive genetic selection pressure in Thai rice blast isolates. The details of sequence variation analysis were described in this article. The information from this study can be used for rice blast resistant breeding program in the future.     

Identification of a new resistance gene Pi-Da(t) from Dacca6 against rice blast fungus (Magnaporthe oryzae) in Jin23B background

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, severely threatens rice production worldwide. A new resistance gene, Pi-Da(t), was found in Dacca6, a local upland rice variety from the Philippines. It was mapped into a region between RM5529 and RM211 on chromosome 2, where no blast resistance gene has been reported, by bulk segregant analysis (BSA) in a BC₁F₂ population from a cross between Dacca6 and Jin23B. The presence of Pi-Da(t) in Jin23B background, an elite parental line preferred for its good grain quality and widely adopted in China??s three-line hybrid rice breeding program over the past 20?years, was verified by BSA and graphical genotyping with additional eight BC₁F₂ bulks. This work presents an example of combining gene mapping work and gene introgression with BSA and graphical genotyping methods in a backcross (BC) breeding scheme. Both the resistant Jin23B line and the linked markers will provide useful information and materials for marker-assisted breeding against blast disease in rice.     

LOX genes in blast fungus (Magnaporthe grisea) resistance in rice

Plant Lipoxygenases (LOX) are known to play major role in plant immunity by providing front-line defense against pathogen-induced injury. To verify this, we isolated a full-length OsLOX3 gene and also 12 OsLOX cDNA clones from Oryza sativa indica (cultivar Pusa Basmati 1). We have examined the role played by LOXs in plant development and during attack by blast pathogen Magnaporthe grisea. Gene expression, promoter region analysis, and biochemical and protein structure analysis of isolated OsLOX3 revealed significant homology with LOX super family. Protein sequence comparison of OsLOXs revealed high levels of homology when compared with japonica rice (up to100%) and Arabidopsis (up to 64%). Isolated LOX3 gene and 12 OsLOX cDNAs contained the catalytic LOX domains much required for oxygen binding and synthesis of oxylipins. Amino acid composition, protein secondary structure, and promoter region analysis (with abundance of motifs CGTCA and TGACG) support the role of OsLOX3 gene in providing resistance to diseases in rice plants. OsLOX3 gene expression analysis of root, shoot, flag leaf, and developing and mature seed revealed organ specific patterns during rice plant development and gave evidence to association between tissue location and physiological roles played by individual OsLOXs. Increased defense activity of oxylipins was observed as demonstrated by PCR amplification of OsLOX3 gene and upon inoculation with virulent strains of M. grisea and ectopic application of methyl jasmonate in the injured leaf tissue in adult rice plants.     

Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus ( Magnaporthe grisea) in transgenic rice

Transgenic rice ( Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.     

Activity profile of defence-related enzymes in rice genotypes (Oryza sativa L.) against rice blast (Magnaporthe oryzae)

The control and infected leaf samples of blast resistant and susceptible rice genotypes were evaluated for activities of defence-related enzymes viz., total phenol content, chitinase, phenylalanine ammonia lyase (PAL), β-glycosidase, antioxidative enzymes, superoxide dismutase, peroxidase and ascorbate peroxidase. The level of total phenol content and the activity profile of chitinase, PAL and β-glycosidase significantly increased in both blast-resistant and susceptible rice genotypes with comparatively higher level induction Tetep, NLR-20104 and Swarnadhan the blast-resistant genotypes. The antioxidative enzymes were comparatively higher in the leaf samples of blast-resistant genotypes recording highest increase in NLR-20104 and KJT-5. The constitutive levels of total phenols and activity of defence-related and antioxidative enzymes in the control leaf samples differed among the genotypes and were even higher in the two blast susceptible genotypes (EK-70 and Chimansal). However, the level of induction as evident from the activity profile differences between control and infected leaf samples suggests higher level of induction was more which is indicative of the induced defence response. The genotype recording maximum induction of defence-related and antioxidative enzymes activity could be useful criteria in screening for blast resistant genotype in rice.     

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase

Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R₀), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines. Received: 16 November 1998 / Accepted: 30 January 1999     

Transformation of the rice blast fungus Magnaporthe grisea to benomyl resistance

A transformation method based on a dominant selectable marker (benomyl resistance) was developed for the rice blast fungus Magnaporthe grisea. The heterologous gene for -tubulin from Neurospora crassa (pBT3) was used to obtain benomyl-resistant M. grisea transformants at a frequency of 20 to 30/g of DNA. Control transformations carried out with a plasmid conferring hygromycin resistance or a derivative of pBT3 containing a repetitive DNA sequence, yielded the same frequency of transformation as that of pBT3. Molecular analysis of the transformants indicated multiple integration of the vector DNA.     

Organization of chromosome ends in the rice blast fungus, Magnaporthe oryzae

Eukaryotic pathogens of humans often evade the immune system by switching the expression of surface proteins encoded by subtelomeric gene families. To determine if plant pathogenic fungi use a similar mechanism to avoid host defenses, we sequenced the 14 chromosome ends of the rice blast pathogen, Magnaporthe oryzae. One telomere is directly joined to ribosomal RNA-encoding genes, at the end of the ~2 Mb rDNA array. Two are attached to chromosome-unique sequences, and the remainder adjoin a distinct subtelomere region, consisting of a telomere-linked RecQ-helicase (TLH) gene flanked by several blocks of tandem repeats. Unlike other microbes, M.oryzae exhibits very little gene amplification in the subtelomere regions—out of 261 predicted genes found within 100 kb of the telomeres, only four were present at more than one chromosome end. Therefore, it seems unlikely that M.oryzae uses switching mechanisms to evade host defenses. Instead, the M.oryzae telomeres have undergone frequent terminal truncation, and there is evidence of extensive ectopic recombination among transposons in these regions. We propose that the M.oryzae chromosome termini play more subtle roles in host adaptation by promoting the loss of terminally-positioned genes that tend to trigger host defenses.     

Mapping of the quantitative trait locus (QTL) conferring partial resistance to rice leaf blast disease

Malaysian rice, Pongsu Seribu 2, has wide-spectrum resistance against blast disease. Chromosomal locations conferring quantitative resistance were detected by linkage mapping with SSRs and quantitative trait locus (QTL) analysis. For the mapping population, 188 F₃ families were derived from a cross between the susceptible cultivar, Mahsuri, and a resistant variety, Pongsu Seribu 2. Partial resistance to leaf blast in the mapping population was assessed. A linkage map covering ten chromosomes and consisting of 63 SSR markers was constructed. 13 QTLs, including 6 putative and 7 putative QTLs, were detected on chromosomes 1, 2, 3, 5, 6, 10, 11 and 12. The resulting phenotypic variation due to a single QTL ranged from 2 to 13 %. These QTLs accounted for approx. 80 % of the total phenotypic variation within the F₃ population. Therefore, partial resistance to blast in Pongsu Seribu 2 is due to combined effects of multiple loci with major and minor effects.     

Genome-wide association mapping of virulence gene in rice blast fungus Magnaporthe oryzae using a genotyping by sequencing approach

Magnaporthe oryzae is a fungal pathogen causing blast disease in many plant species. In this study, seventy three isolates of M. oryzae collected from rice (Oryza sativa) in 1996–2014 were genotyped using a genotyping-by-sequencing approach to detect genetic variation. An association study was performed to identify single nucleotide polymorphisms (SNPs) associated with virulence genes using 831 selected SNP and infection phenotypes on local and improved rice varieties. Population structure analysis revealed eight subpopulations. The division into eight groups was not related to the degree of virulence. Association mapping showed five SNPs associated with fungal virulence on chromosome 1, 2, 3, 4 and 7. The SNP on chromosome 1 was associated with virulence against RD6-Pi7 and IRBL7-M which might be linked to the previously reported AvrPi7.     

Enhanced resistance to the rice blast fungus Magnaporthe grisea conferred by expression of a cecropin A gene in transgenic rice

Cecropins are a family of antimicrobial peptides, which constitute an important key component of the immune response in insects. Here, we demonstrate that transgenic rice (Oryza sativa L.) plants expressing the cecropin A gene from the giant silk moth Hyalophora cecropia show enhanced resistance to Magnaporthe grisea, the causal agent of the rice blast disease. Two plant codon-optimized synthetic cecropin A genes, which were designed either to retain the cecropin A peptide in the endoplasmic reticulum, the ER-CecA gene, or to secrete cecropin A to the extracellular space, the Ap-CecA gene, were prepared. Both cecropin A genes were efficiently expressed in transgenic rice. The inhibitory activity of protein extracts prepared from leaves of cecropin A-expressing plants on the in vitro growth of M. grisea indicated that the cecropin A protein produced by the transgenic rice plants was biologically active. Whereas no effect on plant phenotype was observed in ER-CecA plants, most of the rice lines expressing the Ap-CecA gene were non-fertile. Cecropin A rice plants exhibited resistance to rice blast at various levels. Transgene expression of cecropin A genes was not accompanied by an induction of pathogenesis-related (PR) gene expression supporting that the transgene product itself is directly active against the pathogen. Taken together, the results presented in this study suggest that the cecropin A gene, when designed for retention of cecropin A into the endoplasmic reticulum, could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.