Rice lesion mimic mutants with enhanced resistance to diseases

Lesion mimic mutants are characterized by the formation of necrotic lesions in the absence of pathogens. Such genetic defects often result in enhanced resistance to pathogen infection and constitutive expression of defense response genes. To understand the genetic mechanisms leading to these mutations, we characterized 21 lesion mimic mutants isolated from IR64 rice mutant populations produced by mutagenesis with diepoxybutane (D), gamma rays (G), and fast neutrons (F). Four mutations are controlled by single dominant genes, one of which is inherited maternally. Five lesion mimics are allelic to known spotted leaf (spl) mutants spl1, spl2, spl3, or spl6. In total, 11 new lesion mimic mutations, named spl16, spl17, and spl19 through Spl27, were established based on allelism tests. Two lesion mimics, spl17 and Spl26 showed enhanced resistance to multiple strains of Magnaporthe oryzae, the rice blast pathogen, and Xanthomonas oryzae pv. oryzae, the bacterial blight (BB) pathogen. Co-segregation analyses of blast and BB resistance and lesion mimic phenotypes in segregating populations of spl17 and Spl26 indicate that enhanced resistance to the two diseases is conferred by mutations in the lesion mimic genes. A double mutant produced from two independent lesion mimics showed more severe lesions and higher level of resistance to X. o. pv. oryzae than their single mutant parents indicating a synergistic effect of the two mutations. In mutants that exhibit enhanced disease resistance to both pathogens, increases in expression of defense response genes PR-10a, POX22.3, and PO-C1 were correlated with lesion mimic development and enhancement of resistance. These lesion mimic mutants may provide essential materials for a comprehensive dissection of the disease resistance pathways in rice.     

Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight

Many plant mutants develop spontaneous lesions that resemble disease symptoms in the absence of pathogen attack. In several pathosystems, lesion mimic mutations have been shown to be involved in programmed cell death, which in some instances leads to enhanced disease resistance to multiple pathogens. We investigated the relationship between spontaneous cell death and disease resistance in rice with nine mutants with a range of lesion mimic phenotypes. All nine mutations are controlled by recessive genes and some of these mutants have stunted growth and other abnormal characteristics. The lesion mimics that appeared on the leaves of these mutants were caused by cell death as measured by trypan blue staining. Activation of six defense-related genes was observed in most of the mutants when the mimic lesions developed. Four mutants exhibited significant enhanced resistance to rice blast. One of the mutants, spl11, confers non-race-specific resistance not only to blast but also to bacterial blight. The level of resistance in the spl11 mutant to the two pathogens correlates with the defense-related gene expression and lesion development on the leaves. The results suggest that some lesion mimic mutations in rice may be involved in disease resistance, and cloning of these genes may provide a clue to developing broad-spectrum resistance to diverse pathogens.     

Disruption of gene SPL35, encoding a novel CUE domain‐containing protein,leads to cell death and enhanced disease response in rice

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H₂O₂, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.     

Semi‐dominant mutation in the cysteine‐rich receptor‐like kinase gene,ALS1, conducts constitutive defence response in rice

• Plants have evolved a sophisticated two‐branch defence system to prevent the growth and spread of pathogen infection. The novel Cys‐rich repeat (CRR) containing receptor‐like kinases, known as CRKs, were reported to mediate defence resistance in plants. For rice, there are only two reports of CRKs. A semi‐dominant lesion mimic mutant als1 (apoptosis leaf and sheath 1) in rice was identified to demonstrate spontaneous lesions on the leaf blade and sheath.
• A map‐based cloning strategy was used for fine mapping and cloning of ALS1, which was confirmed to be a typical CRK in rice. Functional studies of ALS1 were conducted, including phylogenetic analysis, expression analysis, subcellular location and blast resistance identification.
• Most pathogenesis‐related (PR) genes and other defence‐related genes were activated and up‐regulated to a high degree. ALS1 was expressed mainly in the leaf blade and sheath, in which further study revealed that ALS1 was present in the vascular bundles. ALS1 was located in the cell membrane of rice protoplasts, and its mutation did not change its subcellular location. Jasmonic acid (JA) and salicylic acid (SA) accumulation were observed in als1, and enhanced blast resistance was also observed.
• The mutation of ALS1 caused a constitutively activated defence response in als1. The results of our study imply that ALS1 participates in a defence response resembling the common SA‐, JA‐ and NH1‐mediated defence responses in rice.

     

国际水稻所水稻新株系的抗性评价

为了进一步扩大我国稻种资源,丰富水稻育种材料,引进了165份国际水稻研究所在非洲进行穿梭育种的水稻新株系,于2011年和2012年在湖北生态条件下进行稻瘟病抗性、白叶枯病抗性和褐飞虱抗性的评价。评价结果表明,在165份新株系中有14份株系在宜昌和恩施2个稻瘟病病圃鉴定均表现抗或中抗稻瘟病,有40份株系同时高抗或抗白叶枯病菌株ZHE173和GD1358,有19份株系抗或中抗褐飞虱,有7份株系同时抗白叶枯病和稻瘟病,有8份株系同时抗白叶枯病和褐飞虱,有1份株系同时中抗稻瘟病、褐飞虱和白叶枯病。部分材料正在作为中间材料用于水稻育种。     

Fine genetic mapping and physical delimitation of the lesion mimic gene <Emphasis Type="Italic">spotted leaf 5</Emphasis> (<Emphasis Type="Italic">spl5</Emphasis>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Spotted leaf 5 (spl5), a lesion mimic mutant, was first identified in rice (Oryza sativa L.) japonica cv. Norin8 in 1978. This mutant exhibits spontaneous disease-like lesions in the absence of any pathogens and resistance to rice blast and bacterial blight; however, the target gene has not yet been isolated. In the present study, we employed a map-based cloning strategy to finely map the spl5 gene. In an initial mapping with 100 F₂ individuals (spl5/spl5) derived from a cross between the spl5 mutant and indica cv. 93-11, the spl5 gene was located in a 3.3-cM region on chromosome 7 using six simple sequence repeat (SSR) markers. In a high-resolution genetic mapping, two F₂ populations with 3,149 individuals (spl5/spl5) were derived from two crosses between spl5 mutant and two indica cvs. 93-11 and Zhefu802 and six sequence-tagged site (STS) markers were newly developed. Finally, the spl5 gene was mapped to a region of 0.048 cM between two markers SSR7 and RM7121. One BAC/PAC contig map covering these markers’ loci and the spl5 gene was constructed through Pairwise BLAST analysis. Our bioinformatics analysis shows that the spl5 gene is located in the 80-kb region between two markers SSR7 and RM7121 with a high average ratio of physical to genetic distance (1.67 Mb/cM) and eighteen candidate genes. The analysis of these candidate genes indicates that the spl5 gene represents a novel class of regulators controlling cell death and resistance response in plants.     

Isolation,fine mapping and expression profiling of a lesion mimic genotype, <Emphasis Type="Italic">spl</Emphasis><Superscript><Emphasis Type="Italic">NF4050</Emphasis>-<Emphasis Type="Italic">8</Emphasis></Superscript> that confers blast resistance in rice

We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different races of Magnaporthe oryzae and identified a lesion mimic mutant, NF4050-8, that showed lesions similar to naturally occurring spl5 mutant and enhanced resistance to all the three blast races tested. Nested modified-AFLP using Tos17-specific primers and southern hybridization experiments of segregating individuals indicated that the lesion mimic phenotype in NF4050-8 is most likely due to a nucleotide change acquired during the culturing process and not due to Tos17 insertion per se. Inheritance and genetic analyses in two japonica × indica populations identified an overlapping genomic region of 13 cM on short arm of chromosome 7 that was linked with the lesion mimic phenotype. High-resolution genetic mapping using 950 F₃ and 3,821 F₄ plants of NF4050-8 × CO39 delimited a 35 kb region flanked by NBARC1 (5.262 Mb) and RM8262 (5.297 Mb), which contained 6 ORFs; 3 of them were ‘resistance gene related’ with typical NBS–LRR signatures. One of them harbored a NB–ARC domain, which had been previously demonstrated to be associated with cell death in animals. Microarray analysis of NF4050-8 revealed significant up-regulation of numerous defense/pathogenesis-related genes and down-regulation of heme peroxidase genes. Real-time PCR analysis of WRKY45 and PR1b genes suggested possible constitutive activation of a defense signaling pathway downstream of salicylic acid but independent of NH1 in these mutant lines of rice.     

Characterisation,screening and selection of Bacillus subtilis isolates for its biocontrol efficiency against major rice diseases

A Soil bacterium, Bacillus subtilis, isolated from the rhizosphere of rice, showed high biocontrol activity against blast, sheath blight and bacterial blight. In the present study, four B. subtilis strains isolated from paddy soil were studied under laboratory, greenhouse and field conditions. Among the four strains assayed, UASP17 gave maximum inhibition of paddy pathogens and was validated under field trials. B. subtilis (UASP17) under different doses and methods of applications was evaluated for two seasons at Agriculture Research Station (UAS, Raichur). UASP17 was effective in reducing the severity of blast (9.00% and 15.57%), bacterial leaf blight (BLB) (5.00% and 6.11%) and sheath blight (11.93% and 4.17%) diseases for two seasons. The application of bioagent also increased the paddy grain yield (61.00 and 64.30?Q/ha) in the two seasons, respectively. Taken together, these results indicate that B. subtilis UASP17 as seedling dip for 30?min (10?mL/L of water) prior to transplanting and 2.50?L/ha foliar application was effective in managing the diseases of paddy.     

Alterations by a defect in a rice G protein α subunit in probenazole and pathogen-induced responses

The rice dwarf1 (d1) mutant, which is deficient in an α subunit (Gα) of heterotrimeric G protein, was used to obtain specific evidence on the functions of Gα protein in defence signalling in rice. Using proteome analysis, a probenazole‐inducible protein (PBZ1) was detected in the cytosolic fraction of leaf blade of the wild type, but not the d1 mutant. After treatment with probenazol, PBZ1 reached maximal levels at 72 h in the wild type but 96 h in the d1 mutant. The induction of PBZ1 by probenazole treatment was inhibited by protein kinase inhibitors. A 48‐kDa putative mitogen‐activated protein kinase (MAPK) and a 55‐kDa putative Ca²⁺‐dependent protein kinase (CDPK) showed lower activities in the cytosolic fraction of the d1 mutant than that of the wild type. The activities of these protein kinases were enhanced at 24 h in the wild type and 48 h in the d1 mutant after probenazole treatment. Although the d1 mutant responded to the rice blast fungus similarly to the wild type, the d1 mutant developed rice blight symptoms earlier than the wild type when infected with Xoo. In addition, the blight symptoms were more severe on the mutant than on the wild type, and wilting was frequently observed in the d1 mutant. Furthermore, induction by the bacterial infection of the 48‐kDa putative MAPK and PBZ1 was delayed by 2 and 4 d, respectively, in the d1 mutant compared with the wild type. These results indicate that the Gα protein plays a role in the induction of PBZ1 and protein kinases by probenazole and Xoo, and suggest that the 48‐kDa putative MAPK may be involved in a signalling pathway for resistance to bacterial infection.     

Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae

Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map‐based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST–OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower‐related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild‐type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blast‐induced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

Marker-assisted breeding of Chinese elite rice cultivar 9311 for disease resistance to rice blast and bacterial blight and tolerance to submergence

Rice (Oryza sativa L.) is the staple food crop for more than half of the world’s population. The development of hybrid rice is a practical approach to increase rice production. However, rice production was frequently affected by biotic and abiotic stresses. Rice blast and bacterial blight are two major diseases in rice growing regions. Rice plantation is also frequently affected by short-term submergence or seasonal floods in wet seasons and drought in dry seasons. The utilization of natural disease resistance (R) genes and stress tolerance genes in rice breeding is the most economic and efficient way to combat or adapt to these biotic and abiotic stresses. Rice cultivar 9311 is widely planted rice variety, either as inbred rice or the paternal line of two-line hybrid rice. Here, we report the pyramiding of rice blast R gene Pi9, bacterial blight R genes Xa21 and Xa27, and submergence tolerance gene Sub1A in 9311 genetic background through backcrossing and marker-assisted selection. The improved rice line, designated as 49311, theoretically possesses 99.2% genetic background of 9311. 49311 and its hybrid rice, GZ63S/49311, conferred disease resistance to rice blast and bacterial blight and showed tolerance to submergence for over 18 days without significant loss of viability. 49311 and its hybrids had similar agronomic traits and grain quality to 9311 and the control hybrid rice, respectively. The development of 49311 provides an improved paternal line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight and tolerance to submergence.     

Mutation in OsLMS, a gene encoding a protein with two double-stranded RNA binding motifs, causes lesion mimic phenotype and early senescence in rice (Oryza sativa L.)

The rice (Oryza sativa L.) lesion mimic and senescence (lms) EMS-mutant, identified in a japonica cultivar Hitomebore, is characterized by a spontaneous lesion mimic phenotype during its vegetative growth, an accelerated senescence after flowering, and enhanced resistance to rice blast (Magnaporthe oryzae). To isolate the OsLMS gene, we crossed the lms mutant to Kasalath (indica), and used mutant F(2) plants to initially map the candidate region to about 322-kb on the long arm of chromosome 2. Illumina whole-genome re-sequencing of the mutant and aligning the reads to Hitomebore reference sequence within the candidate region delineated by linkage analysis identified a G to A nucleotide substitution. The mutation corresponded to the exon-intron splicing junction of a novel gene that encodes a carboxyl-terminal domain (CTD) phosphatase domain and two double stranded RNA binding motifs (dsRBM) containing protein. By PCR amplification, we confirmed that the mutation causes splicing error that is predicted to introduce a premature stop codon. RNA interference (RNAi) transgenic lines with suppressed expression of LMS gene exhibited the lesion mimic phenotype, confirming that the mutation identified in LMS is responsible for the mutant phenotype. OsLMS shares a moderate amino-acid similarity to the Arabidopsis FIERY2/CPL1 gene, which is known to control many plant processes such as stress response and development. Consistence with this similarity, the lms mutant shows sensitivity to cold stress at the early growth stage, suggesting that LMS is a negative regulator of stress response in rice.     

Engineering broad-spectrum disease-resistant rice by editing multiple susceptibility genes

Rice blast and bacterial blight are important diseases of rice (Oryza sativa) caused by the fungus Magnaporthe oryzae and the bacterium Xanthomonas oryzae pv. oryzae (Xoo), respectively. Breeding rice varieties for broad-spectrum resistance is considered the most effective and sustainable approach to controlling both diseases. Although dominant resistance genes have been extensively used in rice breeding and production, generating disease-resistant varieties by altering susceptibility (S) genes that facilitate pathogen compatibility remains unexplored. Here, using CRISPR/Cas9 technology, we generated loss-of-function mutants of the S genes Pi21 and Bsr-d1 and showed that they had increased resistance to M. oryzae. We also generated a knockout mutant of the S gene Xa5 that showed increased resistance to Xoo. Remarkably, a triple mutant of all three S genes had significantly enhanced resistance to both M. oryzae and Xoo. Moreover, the triple mutant was comparable to the wild type in regard to key agronomic traits, including plant height, effective panicle number per plant, grain number per panicle, seed setting rate, and thousand-grain weight. These results demonstrate that the simultaneous editing of multiple S genes is a powerful strategy for generating new rice varieties with broad-spectrum resistance.     

The RhoGAP SPIN6 Associates with SPL11 and OsRac1 and Negatively Regulates Programmed Cell Death and Innate Immunity in Rice

The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.     

新型真菌源激活蛋白诱导水稻抗病性及其生理机制

为明确新型真菌源激活蛋白对水稻抗病性的诱导作用及其生理机制,研究了激活蛋白对水稻稻瘟病和白叶枯病的诱导抗病性,监测了激活蛋白处理后水稻过氧化物酶（POD）、多酚氧化酶（PPO）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）活性及过氧化氢（H₂O₂）含量变化。结果表明,1～6 μg·mL^-1激活蛋白对稻瘟病和白叶枯病的诱抗效果分别为45.2%～71.4%和47.6%～66.3%,以6 μg·mL^-1激活蛋白的诱抗效果最好。与对照相比,2 μg·mL^-1激活蛋白处理水稻后3～15 d内不同程度诱导了防御酶POD、PPO和SOD活性,抑制CAT活性,提高H₂O₂含量。新型真菌源激活蛋白能够诱导水稻产生对稻瘟病和白叶枯病的抗病性,其诱导抗性机制与水稻体内的活性氧代谢密切相关。     

OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide,jasmonic acid,and phytoalexin metabolic pathways

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H₂O₂) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H₂O₂ in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.