Blast Resistance Gene Research Articles

Weighted Gene Co-Expression Network Coupled with a Critical-Time-Point Analysis during Pathogenesis for Predicting the Molecular Mechanism Underlying Blast Resistance in Rice

BackgroundRice blast, caused by the ascomycete fungus M. oryzae, is one of the most important diseases of rice. Although many blast resistance (R) genes have been identified and deployed in rice varieties, the molecular mechanisms responsible for the R gene-mediated defense responses are yet not fully understood.ResultsIn this study, we used comparative transcriptomic analysis to explore the molecular mechanism involved in Piz-t-mediated resistance in a transgenic line containing Piz-t (NPB-Piz-t) compared to Nipponbare (NPB). Clustering and principal component analysis (PCA) revealed that the time-point at 24-h post inoculation (hpi) was the most important factor distinguishing the four time-points, which consisted of four genes of mitogen-activated protein kinases (MAPKs) signaling pathway, one gene related to WRKY DNA-binding domain containing protein, five pathogenesis-related protein (OsPR1s) genes, and three genes of R proteins involving in the most significant protein-protein interaction (PPI) pathway. Using weighted gene co-expression network analysis (WGCNA) to investigate RNA-seq data across 0, 24, 48, and 72 hpi, nine modules with similar patterns expression pattern (SEP) and three modules with differential expression pattern (DEP) between NPB-Piz-t and NPB across 0, 24, 48, and 72 hpi with KJ201 (referred to as Piz-t-KJ201 and NPB-KJ201) were identified. Among these the most representative SEP green-yellow module is associated with photosynthesis, and DEP pink module comprised of two specific expressed nucleotide-binding domain and leucine-rich repeat (NLR) genes of LOC_Os06g17900 and LOC_Os06g17920 of Pi2/9 homologous, three NLR genes of LOC_Os11g11810, LOC_Os11g11770, and LOC_Os11g11920 which are putatively associated with important agronomic traits, and a B3 DNA binding domain containing protein related genes (LOC_Os10g39190). Knockout of LOC_Os10g39190 via CRISPR-Cas9 resulted in plant death at the seedling stage.ConclusionsThe research suggested that Piz-t and multiple NLR network might play important roles in the regulation of the resistance response in the Piz-t-KJ201 interaction system. The identified genes provide an NLR repository to study the rice-M. oryzae interaction system and facilitate the breeding of blast-resistant cultivars in the future.

Reduction of cadmium accumulation in the grains of male sterile rice Chuang-5S carrying Pi48 or Pi49 through marker-assisted selection.

To reduce cadmium (Cd) accumulation in the grains of rice Chuang-5S (C5S), a gene OsHMA3 and a QTL qlGCd3 related to low-Cd accumulation were separately introgressed into the recipient parent C5S (male sterile line) by molecular marker-assisted breeding. The recurrent parent C5S was then replaced by NIL (near-isogenic line)-C5S with the blast resistance gene Pi48 or Pi49 to construct the BC2F1 generation. Finally, two groups of improved materials of C5S, which pyramided the gene/QTL associated with low-Cd accumulation and blast resistance gene, were developed. The Cd accumulation, agronomic traits, genetic background and blast resistance of these improved C5S materials were evaluated. The results showed that the average Cd content of improved C5S material carrying OsHMA3 and qlGCd3 was, respectively, reduced by 52.8% and 50.8% compared with that of C5S, indicating that the gene related to low-Cd accumulation was, successfully, transferred to C5S with stable expression. The main agronomic traits of the improved materials were consistent with those of C5S. Besides, the improved C5S lines showed stronger blast resistance than C5S and more than 88% similarity to the genetic background of C5S. These two groups of improved materials may be further utilized for the breeding of advanced male sterile lines or superior hybrid rice.

Rice Breeding in Russia Using Genetic Markers.

The article concentrates on studying tolerance to soil salinization, water flooding, and blast in Russian and Asian rice varieties, as well as hybrids of the second and third generations from their crossing in order to obtain sustainable paddy crops based on domestic varieties using DNA markers. Samples IR 52713-2B-8-2B-1-2, IR 74099-3R-3-3, and NSIC Rc 106 were used as donors of the SalTol tolerance gene. Varieties with the Sub1A locus were used as donors of the flood resistance gene: Br-11, CR-1009, Inbara-3, TDK-1, and Khan Dan. The lines C101-A-51 (Pi-2), C101-Lac (Pi-1, Pi-33), IR-58 (Pi-ta), and Moroberekan (Pi-b) were used to transfer blast resistance genes. Hybridization of the stress-sensitive domestic varieties Novator, Flagman, Virazh, and Boyarin with donor lines of the genes of interest was carried out. As a result of the studies carried out using molecular marking based on PCR in combination with traditional breeding, early-maturing rice lines with genes for resistance to salinity (SalTol) and flooding (Sub1A), suitable for cultivation in southern Russia, were obtained. Introgression and pyramiding of the blast resistance genes Pi-1, Pi-2, Pi-33, Pi-ta, and Pi-b into the genotypes of domestic rice varieties were carried out. DNA marker analysis revealed disease-resistant rice samples carrying 5 target genes in a homozygous state. The created rice varieties that carry the genes for blast resistance (Pentagen, Magnate, Pirouette, Argamac, Kapitan, and Lenaris) were submitted for state variety testing. The introduction of such varieties into production will allow us to avoid epiphytotic development of the disease, preserving the biological productivity of rice and obtaining environmentally friendly agricultural products.

Evaluation of durability of blast resistance gene Rmg8 in common wheat based on analyses of its corresponding avirulence gene

Wheat blast caused by the Triticum pathotype of Pyricularia oryzae is a serious threat to wheat production in South America and Asia. Rmg8 is a promising gene for resistance to the wheat blast fungus found in common wheat. To predict its durability, stability of its corresponding avirulence gene, AVR-Rmg8, was evaluated based on the genome structure around AVR-Rmg8 in an isolate from Triticum and on polymorphisms of AVR-Rmg8 in isolates from Lolium spp., which are closely related to Triticum isolates. AVR-Rmg8 was located in a gene-rich, repeat-poor region, suggesting that AVR-Rmg8 is not as easily lost as avirulence genes located in repeat-rich regions. This finding implies that Rmg8 is relatively durable compared with resistance genes corresponding to avirulence genes in repeat-rich regions. AVR-Rmg8 was widely distributed in Lolium isolates and comprised three types or variants, eL1, eL2, and eL3. Complementation tests revealed that eL1 and eL2 were functional and recognized by Rmg8. In eL3, however, retrotransposon Pyret was inserted into the ORF of AVR-Rmg8. Actually, three isolates carrying eL3 were virulent on common wheat cultivar S-615 carrying Rmg8, indicating that the insertion of Pyret caused a loss of function of AVR-Rmg8. Nevertheless, the three isolates have survived in nature, suggesting that AVR-Rmg8 is not indispensable for survival of Pyricularia isolates and, in turn, that Rmg8 is not likely to be completely durable.

Transcriptome analysis of xa5-mediated resistance to bacterial leaf streak in rice (Oryza sativa L.)

Bacterial leaf steak (BLS) caused by Xanthomonas oryzae pv. oryzicola (Xoc) is a devastating disease in rice production. The resistance to BLS in rice is a quantitatively inherited trait, of which the molecular mechanism is still unclear. It has been proved that xa5, a recessive bacterial blast resistance gene, is the most possible candidate gene of the QTL qBlsr5a for BLS resistance. To study the molecular mechanism of xa5 function in BLS resistance, we created transgenic lines with RNAi of Xa5 (LOC_Os05g01710) and used RNA-seq to analyze the transcriptomes of a Xa5-RNAi line and the wild-type line at 9 h after inoculation with Xoc, with the mock inoculation as control. We found that Xa5-RNAi could (1) increase the resistance to BLS as expected from xa5; (2) alter (mainly up-regulate) the expression of hundreds of genes, most of which were related to disease resistance; and (3) greatly enhance the response of thousands of genes to Xoc infection, especially of the genes involved in cell death pathways. The results suggest that xa5 is the cause of BLS-resistance of QTL qBlsr5a and it displays BLS resistance effect probably mainly because of the enhanced response of the cell death-related genes to Xoc infection.

Genome wide association studies for japonica rice resistance to blast in field and controlled conditions

BackgroundRice blast, caused by the fungus Pyricularia oryzae, represents the most damaging fungal disease of rice worldwide. Utilization of rice resistant cultivars represents a practical way to control the disease. Most of the rice varieties cultivated in Europe and several other temperate regions are severely depleted of blast resistance genes, making the identification of resistant sources in genetic background adapted to temperate environments a priority. Given these assumptions, a Genome Wide Association Study (GWAS) for rice blast resistance was undertaken using a panel of 311 temperate/tropical japonica and indica accessions adapted to temperate conditions and genotyped with 37,423 SNP markers. The panel was evaluated for blast resistance in field, under the pressure of the natural blast population, and in growth chamber, using a mixture of three different fungal strains.ResultsThe parallel screening identified 11 accessions showing high levels of resistance in the two conditions, representing potential donors of resistance sources harbored in rice genotypes adapted to temperate conditions. A general higher resistance level was observed in tropical japonica and indica with respect to temperate japonica varieties. The GWAS identified 14 Marker-Traits Associations (MTAs), 8 of which discovered under field conditions and 6 under growth chamber screening. Three MTAs were identified in both conditions; five MTAs were specifically detected under field conditions while three for the growth chamber inoculation. Comparative analysis of physical/genetic positions of the MTAs showed that most of them were positionally-related with cloned or mapped blast resistance genes or with candidate genes whose functions were compatible for conferring pathogen resistance. However, for three MTAs, indicated as BRF10, BRF11–2 and BRGC11–3, no obvious candidate genes or positional relationships with blast resistance QTLs were identified, raising the possibility that they represent new sources of blast resistance.ConclusionsWe identified 14 MTAs for blast resistance using both field and growth chamber screenings. A total of 11 accessions showing high levels of resistance in both conditions were discovered. Combinations of loci conferring blast resistance were identified in rice accessions adapted to temperate conditions, thus allowing the genetic dissection of affordable resistances present in the panel. The obtained information will provide useful bases for both resistance breeding and further characterization of the highlighted resistance loci.

Effectiveness of the Wheat Blast Resistance Gene Rmg8 in Bangladesh Suggested by Distribution of an AVR-Rmg8 Allele in the Pyricularia oryzae Population.

Wheat blast caused by the Triticum pathotype of Pyricularia oryzae was first reported in 1985 in Brazil and recently spread to Bangladesh. We tested whether Rmg8 and RmgGR119, recently identified resistance genes, were effective against Bangladeshi isolates of the pathogen. Common wheat accessions carrying Rmg8 alone (IL191) or both Rmg8 and RmgGR119 (GR119) were inoculated with Brazilian isolates (Br48, Br5, and Br116.5) and Bangladeshi isolates (T-108 and T-109). Br48, T-108, and T-109 carried the eI type of AVR-Rmg8 (the avirulence gene corresponding to Rmg8) while Br5 and Br116.5 carried its variants, eII and eII' types, respectively. Detached primary leaves of IL191 and GR119 were resistant to all isolates at 25°C. At a higher temperature (28°C), their resistance was still effective against the eI carriers but was reduced to a low level against the eII/eII' carriers. A survey of databases and sequence analyses revealed that all Bangladeshi isolates carried the eI type which induced a higher level of resistance than the eII/eII' types. The resistance of IL191 (Rmg8/-) to the eI carriers was maintained even at the heading stage and at the higher temperature. In addition, GR119 (Rmg8/RmgGR119) displayed higher levels of resistance than IL191 at this stage. These results suggest that Rmg8 combined with RmgGR119 will be useful in breeding for resistance against wheat blast in Bangladesh.

Dissecting the genetic basis of wheat blast resistance in the Brazilian wheat cultivar BR 18-Terena

BackgroundWheat blast, caused by Magnaporthe oryzae Triticum (MoT) pathotype, is a global threat to wheat (Triticum aestivum L.) production. Few blast resistance (R) genes have been identified to date, therefore assessing potential sources of resistance in wheat is important. The Brazilian wheat cultivar BR 18-Terena is considered one of the best sources of resistance to blast and has been widely used in Brazilian breeding programmes, however the underlying genetics of this resistance are unknown.ResultsBR 18-Terena was used as the common parent in the development of two recombinant inbred line (RIL) F6 populations with the Brazilian cultivars Anahuac 75 and BRS 179. Populations were phenotyped for resistance at the seedling and heading stage using the sequenced MoT isolate BR32, with transgressive segregation being observed. Genetic maps containing 1779 and 1318 markers, were produced for the Anahuac 75 × BR 18-Terena and BR 18-Terena × BRS 179 populations, respectively. Five quantitative trait loci (QTL) associated with seedling resistance, on chromosomes 2B, 4B (2 QTL), 5A and 6A, were identified, as were four QTL associated with heading stage resistance (1A, 2B, 4A and 5A). Seedling and heading stage QTL did not co-locate, despite a significant positive correlation between these traits, indicating that resistance at these developmental stages is likely to be controlled by different genes. BR 18-Terena provided the resistant allele for six QTL, at both developmental stages, with the largest phenotypic effect conferred by a QTL being 24.8% suggesting that BR 18-Terena possesses quantitative resistance. Haplotype analysis of 100 Brazilian wheat cultivars indicates that 11.0% of cultivars already possess a BR 18-Terena-like haplotype for more than one of the identified heading stage QTL.ConclusionsThis study suggests that BR 18-Terena possesses quantitative resistance to wheat blast, with nine QTL associated with resistance at either the seedling or heading stage being detected. Wheat blast resistance is also largely tissue-specific. Identification of durable quantitative resistances which can be combined with race-specific R gene-mediated resistance is critical to effectively control wheat blast. Collectively, this work facilitates marker-assisted selection to develop new varieties for cultivation in regions at risk from this emerging disease.

Identification and Fine Mapping of Pi69(t), a New Gene Conferring Broad-Spectrum Resistance Against Magnaporthe oryzae From Oryza glaberrima Steud.

The discovery and deployment of new broad-spectrum resistance (R) genes from cultivated rice and its wild relatives is a strategy to broaden the genetic basis of modern rice cultivars to combat rice blast disease. Oryza glaberrima possessing many valuable traits for tolerance to biotic and abiotic stresses, is an elite gene pool for improvement of Asian cultivated rice. An introgression line IL106 derived from O. glaberrima (Acc. IRGC100137) confers complete resistance to Magnaporthe oryzae in blast nursery. Genetic analysis using 2185 BC6F2 progenies derived from a cross between IL106 and the recurrent parent Dianjingyou 1 showed that IL106 harbors a single dominant resistance gene against M. oryzae strain 09BSH-10-5A. This gene was preliminarily mapped on the long arm of chromosome 6 of rice in a region of ca. 0.9 cM delimited by two SSR markers (RM20650 and RM20701). In order to finely map this gene, 17,100 additional progenies were further analyzed. As a result, this gene was further narrowed down to a region flanked by two molecular markers STS69-15 and STS69-7, and co-segregated with 3 molecular markers, RM20676, STS69-21 and STS69-22 on the long arm of chromosome 6. Based on reference genome sequences, this R gene was mapped in silico in 76.1-Kb and 67.7-Kb physical intervals, and containing 4 and 3 NBS-LRR candidate genes in O. sativa cultivar Nipponbare and O. glaberrima cultivar CG14, respectively. Because no blast resistance gene was finely mapped in this physical interval before, this R gene was considered as not described yet and designated as Pi69(t), which is the first identified and finely mapped blast R gene from O. glaberrima, as far as we know. Evaluation of IL106 with 151 blast strains collected from 6 countries in Asia showed that 148 strains are avirulent on IL106, suggesting that Pi69(t) is a broad-spectrum blast R gene, and a promising resistant resource for improvement of Asian cultivated rice.

Molecular analysis of five rice blast resistant genes in landraces from Myanmar and Laos

AbstractIn order to understand rice blast resistance gene patterns in rice landraces from Myanmar and Laos, we analysed thePita,Pib,Pikh,Pi9andPi5genes in 80 landraces through the use of functional molecular markers and functional fragment sequencing. These landraces were separated into four haplotypes (H1–H4) based upon three mutations identified in thePitasequence. All four haplotypes were detected among landraces from Myanmar, whereas only the H1 and H2 haplotypes were detected among landraces from Laos. We additionally grouped landraces carrying 0–4 resistance genes into 18 genotypes based upon their resistance-susceptibility patterns and found that 16 of these genotypes were detected among Myanmar landraces at relative frequencies ranging from 2.50–12.50%, while 10 were detected among landraces from Laos at relative frequencies of 2.50–42.50%. The ‘Pib( + )pikh(−)pi9(−)pi5(−)pita(−)’ genotype was found to be dominant, accounting for 12.5 and 42.5% of landraces from Myanmar and Laos, respectively. ThePib,Pikh,Pi5andPitagenes were detected in 52.50, 32.50, 37.50 and 30.00% of landraces from Myanmar respectively, whereas they were detected in 52.50, 12.50, 30.00 and 17.50% of landraces from Laos, respectively. ThePi9gene was rarely detected among analysed landraces and was particularly rare in those from Laos. These findings suggest that rice landraces from Myanmar are more genetically diverse than those from Laos, with clear differences in blast resistance gene patterns between landraces from these two countries.

Molecular characterization of popular rice (Oryza sativa L.) varieties of India and association analysis for blast resistance

Blast is a major biotic factor accountable for near-complete yield loss in rice (Oryza sativa L.). Thirty-seven leaf blast resistant lines were found out of 103 rice germplasm through phenotyping. Subsequent genotyping of the germplasm for five important blast resistance genes of India namely, Pitp, Pi1, Pi2, Pi9, and Pi54 using gene-specific markers revealed the prevalence of Pitp as more and Pi2 as least in the tested lines. An initial genetic diversity study showed the highest PIC value of 0.77 for RM234 defining its high polymorphic behaviour. Model-based and distance-based approaches showed a high correlation in displaying genetic diversity. The model-based structural analysis based on log-likelihood (∆k) displayed three clusters and remaining accessions under admixture. Distance-based analysis using Unweighted Pair Group Method with Arithmetic Mean method also divided the accessions into three major clusters. Analysis of molecular variance revealed a variation of 19% among the populations, while 81% within the populations. The association analysis using 101 SSR markers revealed ten newly identified marker-trait associations by General Linear Model and two marker-trait associations using Mixed Linear Model approach. These markers can be validated in a large set of germplasm and can be used in marker-assisted selection. The identified resistant sources having known genes can be readily used in the breeding programmes.

Role of the rice blast resistance gene Pi‐cd in rice (Oryza sativa) breeding programmes

AbstractA series of DNA markers for various agronomic traits may accelerate the success of marker‐assisted selection in practical plant breeding programmes. Here, we developed DNA markers for the blast resistance gene Pi‐cd. In this study, we examined the effects of the Pi‐cd locus on not only blast resistance but also agronomic traits in agriculture. We developed three pyramiding lines (PLs) coupling Pi‐cd with three blast resistance genes, pi21, Pi35 and Pi39. The effect of Pi‐cd on blast resistance was dependent on the coupled resistance genes. Then, we evaluated the effects of Pi‐cd on 13 agronomic traits. Amylose content and 1,000‐grain weight showed significant differences between the PLs and current commercial varieties, which had no negative effects on agronomic trait values. Furthermore, we investigated the distribution of genotype for the Pi‐cd locus among varieties of upland rice. The KT genotype specific to rice blast resistance may be predominant in the varieties. The results suggested that Pi‐cd has the potential to be useful for improving blast resistance in rice breeding programmes.

Marker Assisted Gene Pyramiding (MAGP) for bacterial blight and blast resistance into mega rice variety "Tellahamsa".

Bacterial blight (BB) and fungal blast diseases are the major biotic constraints that limit rice productivity. To sustain yield improvement in rice, it is necessary to developed yield potential of the rice varieties by incorporation of biotic stress resistance genes. Tellahamsa is a well-adapted popular high yielding rice variety in Telangana state, India. However, the variety is highly susceptible to BB and blast. In this study, simultaneous stepwise transfer of genes through marker-assisted backcross breeding (MABB) strategy was used to introgress two major BB (Xa21 and xa13) and two major blast resistance genes (Pi54 and Pi1) into Tellahamsa. In each generation (from F1 to ICF3) foreground selection was done using gene-specific markers viz., pTA248 (Xa21), xa13prom (xa13), Pi54MAS (Pi54) and RM224 (Pi1). Two independent BC2F1 lines of Tellahamsa/ISM (Cross-I) and Tellahamsa/NLR145 (Cross-II) possessing 92% and 94% recurrent parent genome (RPG) respectively were intercrossed to develop ICF1—ICF3 generations. These gene pyramided lines were evaluated for key agro-morphological traits, quality, and resistance against blast at three different hotspot locations as well as BB at two locations. Two ICF3 gene pyramided lines viz., TH-625-159 and TH-625-491 possessing four genes exhibited a high level of resistance to BB and blast. In the future, these improved Tellahamsa lines could be developed as mega varieties for different agro-climatic zones and also as potential donors for different pre-breeding rice research.

Molecular profiling of blast resistance genes and evaluation of leaf and neck blast disease reaction in rice

In the present investigation, phenotypic evaluation of blast disease reaction was conducted at Ponnampet and Mandya districts of Karnataka, India, which indicated that the rice varieties such as IR64, Jaya, KMP153, IR30864, Mandya Sona-1, Mandya Sona-2, KCP-1, Dodda Byra, and Malgudi Sanna were susceptible to both leaf and neck blasts. Further, the rice varieties that were resistant to leaf blast such as KMP200, DHMAS70Q164-1b, Karibatta, Coimbatore Sanna and others showed susceptible reaction to neck blast only. In contrast, the varieties such as JyothixBR2655, Punkutt Kodi, Sirsi, 222 and Gangadale which were resistant to neck blast were found to be susceptible to leaf blast also. Only one variety, BR2655 showed resistance to both leaf and neck blast diseases. The genotypic studies using simple sequence repeat markers showed that the analysis of the distribution of resistance genes and genotyping of the selected rice varieties, and traditional rice varieties from different ecological regions with allele specific markers helped to identify 20 major blast resistance genes. The individual gene frequencies of the 20 major rice blast resistance genes varied from 10.34 to 100%. Less and more frequency of resistance gene distribution occurred in Pi9 and Pizt gene, respectively. The result of this study would help to create strategies for improving rice blast resistance through genetic studies and plant-pathogen interaction.

Identification of Blast Resistance QTLs Based on Two Advanced Backcross Populations in Rice

BackgroundRice blast is an economically important and mutable disease of rice. Using host resistance gene to breed resistant varieties has been proven to be the most effective and economical method to control rice blast and new resistance genes or quantitative trait loci (QTLs) are then needed.ResultsIn this study, we constructed two advanced backcross population to mapping blast resistance QTLs. CR071 and QingGuAi3 were as the donor parent to establish two BC3F1 and derived BC3F2 backcross population in the Jin23B background. By challenging the two populations with natural infection in 2011 and 2012, 16 and 13 blast resistance QTLs were identified in Jin23B/CR071 and Jin23B/QingGuAi3 population, respectively. Among Jin23B/CR071 population, 3 major and 13 minor QTLs have explained the phenotypic variation from 3.50% to 34.08% in 2 years. And, among Jin23B/QingGuAi3 population, 2 major and 11 minor QTLs have explained the phenotypic variation from 2.42% to 28.95% in 2 years.ConclusionsSixteen and thirteen blast resistance QTLs were identified in Jin23B/CR071 and Jin23B/QingGuAi3 population, respectively. QTL effect analyses suggested that major and minor QTLs interaction is the genetic basis for durable blast resistance in rice variety CR071 and QingGuAi3.

Transcriptome Comparison of Defense Responses in the Rice Variety 'Jao Hom Nin' Regarding Two Blast Resistant Genes, Pish and Pik.

Jao Hom Nin (JHN) is a Thai rice variety with broad-spectrum resistant against rice blast fungus. JHN contains two rice blast resistant genes, Pish and Pik, located on chromosome 1 and on chromosome 11, respectively. To understand the blast resistance in JHN, the study of the defense mechanism related to the Pish and Pik genes is crucial. This study aimed to dissect defense response genes between the Pish and Pik genes using the RNA-seq technique. Differentially expressed genes (DEGs) of Pish and Pik backcross inbred lines were identified between 0 and 24 h after inoculation with rice blast spore suspension. The results showed that 1248 and 858 DEGs were unique to the Pish and Pik lines, respectively. The wall-associated kinase gene was unique to the Pish line and the zinc-finger-containing protein gene was unique to the Pik line. Pathogenicity-related proteins PR-4 and PR-10 were commonly found in both Pish and Pik lines. Moreover, DEGs functionally categorized in brassinosteriod, jasmonic acid, and salicylic acid pathways were detected in both Pish and Pik lines. These unique and shared genes in the Pish and Pik rice blast defense responses will help to dissect the mechanisms of plant defense and facilitate rice blast breeding programs.

High nucleotide sequence variation of avirulent gene, AVR-Pita1, in Thai rice blast fungus population

Rice blast disease, caused by Magnaporthe oryzae, is one of the most importance diseases of rice production worldwide. The keyrole of defense mechanism to combat this fungus in rice follows the gene-for-gene concept, which a plant resistant (R) gene product recognizes a fungal avirulent (AVR) effector and triggers the hypersensitive response. However, the AVR genes have been shown to be rapidly evolving resulting in high level of genetic diversity. The aims of this study were to examine the nucleotide sequence variation of AVR-Pita1 gene in Thai rice blast isolates and to identify the severity of blast disease using isogenic line of Pita gene. Seventy-six rice blast isolates collected from different parts of Thailand were used. Gene specific primers for AVR-Pita1 gene coding sequence were designed and used for identifying the genetic diversity of AVR-Pita1 gene by PCR amplification and sequencing. The obtained sequences were analysed for genetic variation and genetic relationship. Our results revealed the association between the sequence variations of AVR-Pita1 and selective forces from Pita gene. This phenomenon demonstrated the coevolution between rice blast resistant gene in rice and avirulent gene in blast fungus. The information about variation and evolutionary mechanisms of AVR gene obtained from this study can be used in rice blast resistant breeding programme.

Blast resistance gene Pi54 over-expressed in rice to understand its cellular and sub-cellular localization and response to different pathogens

Rice blast resistance gene, Pi54 provides broad-spectrum resistance against different strains of Magnaporthe oryzae. Understanding the cellular localization of Pi54 protein is an essential step towards deciphering its place of interaction with the cognate Avr-gene. In this study, we investigated the sub-cellular localization of Pi54 with Green Fluorescent Protein (GFP) as a molecular tag through transient and stable expression in onion epidermal cells (Allium cepa) and susceptible japonica cultivar rice Taipei 309 (TP309), respectively. Confocal microscopy based observations of the onion epidermal cells revealed nucleus and cytoplasm specific GFP signals. In the stable transformed rice plants, GFP signal was recorded in the stomata, upper epidermal cells, mesophyll cells, vascular bundle, and walls of bundle sheath and bulliform cells of leaf tissues. These observations were further confirmed by Immunocytochemical studies. Using GFP specific antibodies, it was found that there was sufficient aggregation of GFP::Pi54protein in the cytoplasm of the leaf mesophyll cells and periphery of the epidermal cells. Interestingly, the transgenic lines developed in this study could show a moderate level of resistance to Xanthomonas oryzae and Rhizoctonia solani, the causal agents of the rice bacterial blight and sheath blight diseases, respectively. This study is a first detailed report, which emphasizes the cellular and subcellular distribution of the broad spectrum blast resistance gene Pi54 in rice and the impact of its constitutive expression towards resistance against other fungal and bacterial pathogens of rice.

Bulked Segregant Analysis Coupled with Whole-Genome Sequencing (BSA-Seq) Mapping Identifies a Novel pi21 Haplotype Conferring Basal Resistance to Rice Blast Disease.

Basal or partial resistance has been considered race-non-specific and broad-spectrum. Therefore, the identification of genes or quantitative trait loci (QTLs) conferring basal resistance and germplasm containing them is of significance in breeding crops with durable resistance. In this study, we performed a bulked segregant analysis coupled with whole-genome sequencing (BSA-seq) to identify QTLs controlling basal resistance to blast disease in an F2 population derived from two rice varieties, 02428 and LiXinGeng (LXG), which differ significantly in basal resistance to rice blast. Four candidate QTLs, qBBR-4, qBBR-7, qBBR-8, and qBBR-11, were mapped on chromosomes 4, 7, 8, and 11, respectively. Allelic and genotypic association analyses identified a novel haplotype of the durable blast resistance gene pi21 carrying double deletions of 30 bp and 33 bp in 02428 (pi21-2428) as a candidate gene of qBBR-4. We further assessed haplotypes of Pi21 in 325 rice accessions, and identified 11 haplotypes among the accessions, of which eight were novel types. While the resistant pi21 gene was found only in japonica before, three Chinese indica varieties, ShuHui881, Yong4, and ZhengDa4Hao, were detected carrying the resistant pi21-2428 allele. The pi21-2428 allele and pi21-2428-containing rice germplasm, thus, provide valuable resources for breeding rice varieties, especially indica rice varieties, with durable resistance to blast disease. Our results also lay the foundation for further identification and functional characterization of the other three QTLs to better understand the molecular mechanisms underlying rice basal resistance to blast disease.

The broad-spectrum rice blast resistance (R) gene Pita2 encodes a novel R protein unique from Pita

BackgroundRice blast is generally considered the most devastating rice disease worldwide. The development of resistant varieties has been proven to be the most economical strategy to control the disease. A cluster of resistant (R) genes on rice chromosome 12 including Pita, Pita2 and Ptr has been studies for decades. However, the relationship between these R genes has not been well established.ResultsIn this study, we compared the resistance spectra controlled by Pita2 and Pita by testing their monogenic lines (MLs) in four hotspots found in the Philippines and Burundi from 2014 to 2018. The reaction patterns were distinct in two countries and that Pita2-mediated field resistance was relatively prevalent. Pathogenicity tests using 328 single-spore isolates in greenhouse further verified that IRBLta2-Re for Pita2 conferred a relatively broader spectrum resistance than those of Pita. Rough and fine mapping of Pita2 were conducted using F2 and F3 populations derived from IRBLta2-Re [CO] and CO 39 consisting of 4344 progeny to delimit Pita2 in a genomic interval flanked by two markers 12 g18530 and 12 g18920 proximal to the centromere of chromosome 12. Alignment of the markers to the genomic sequence of IR64, which harbors Pita2 verified by genetic analysis, approximately delimited the candidate gene(s) within 313-kb genomic fragment. The two Pita2 suppressive mutants that contain mutations within Pita2 were verified and identified. Comparative sequence analysis in these two mutants further identified that each individual allele contains a single nucleotide substitution at a different position resulting in nonsense and missense mutations in the protein product of LOC_Os12g18729. On the contrary, no sequence mutation was detected in other candidate genes, indicating that mutations in LOC_Os12g18729 were responsible for the loss of function of Pita2. Pita2 encodes a novel R protein unique from Pita, which is exactly identical to the previously cloned Ptr. Moreover, based on the resistance gene analysis of rice varieties and mutants containing Pita, it was found that Pita2 rather than Pita was responsible for the specificity to some differential isolates with AvrPita. The diagnosis and survey of Pita2 in IRRI released varieties showed relatively low frequency, implying a high value of its application for breeding resistant varieties against rice blast via marker assisted selection.ConclusionOur study clarified the relationship between Pita, Pita2 and Ptr. Pita2 is identical to Ptr and distinct from Pita in both sequence and chromosomal location although Pita2 and Pita are genetically linked to each other. The loss of function of Pita2 but not Pita eliminate the specificity to some AvrPita containing isolates, however, the mechanism underlying the recognition between Pita2/Pita and AvrPita remains elusive.