Characterization and genetic analysis of a light- and temperature-sensitive spotted-leaf mutant in rice

A rice spotted-leaf mutant was isolated from an ethane methyl sulfonate (EMS) -induced IR64 mutant bank. The mutant, designated as spl30 (spotted-leaf30), displayed normal green leaf color under shade but exhibited red-brown lesions under natural summer field conditions. Initiation of the lesions was induced by light and the symptom was enhanced at 33 (°) C relative to 26 (°) C. Histochemical staining did not show cell death around the red-brown lesions. Chlorophyll contents in the mutant were significantly lower than those of the wild type while the ratio of chlorophyll a/b remained the same, indicating that spl30 was impaired in biosynthesis or degradation of chlorophyll. Disease reaction patterns of the mutant to Xanthomonas oryzae pv. oryzae were largely unchanged to most races tested except for a few strains. Genetic analysis showed that the mutation was controlled by a single recessive gene, tentatively named spl30(t), which co-segregated with RM15380 on chromosome 3, and was delimited to a 94 kb region between RM15380 and RM15383. Spl30(t) is likely a novel rice spotted-leaf gene since no other similar genes have been identified near the chromosomal region. The genetic data and recombination populations provided in this study will enable further fine-mapping and cloning of the gene.     

Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence

Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including innate immune responses in plants. However, the mechanism of the E3 ligase involvement in plant innate immunity is unclear. We report that a rice gene, OsBBI1, encoding a RING finger protein with E3 ligase activity, mediates broad-spectrum disease resistance. The expression of OsBBI1 was induced by rice blast fungus Magnaporthe oryzae, as well as chemical inducers, benzothiadiazole and salicylic acid. Biochemical analysis revealed that OsBBI1 protein possesses E3 ubiquitin ligase activity in vitro. Genetic analysis revealed that the loss of OsBBI1 function in a Tos17-insertion line increased susceptibility, while the overexpression of OsBBI1 in transgenic plants conferred enhanced resistance to multiple races of M. oryzae. This indicates that OsBBI1 modulates broad-spectrum resistance against the blast fungus. The OsBBI1-overexpressing plants showed higher levels of H(2)O(2) accumulation in cells and higher levels of phenolic compounds and cross-linking of proteins in cell walls at infection sites by M. oryzae compared with wild-type (WT) plants. The cell walls were thicker in the OsBBI1-overexpressing plants and thinner in the mutant plants than in the WT plants. Our results suggest that OsBBI1 modulates broad-spectrum resistance to blast fungus by modifying cell wall defence responses. The functional characterization of OsBBI1 provides insight into the E3 ligase-mediated innate immunity, and a practical tool for constructing broad-spectrum resistance against the most destructive disease in rice.     

Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility

The key regulator of salicylic acid (SA)-mediated resistance, NPR1, is functionally conserved in diverse plant species, including rice (Oryza sativa L.). Investigation in depth is needed to provide an understanding of NPR1-mediated resistance and a practical strategy for the improvement of disease resistance in the model crop rice. The rice genome contains five NPR1-like genes. In our study, three rice homologous genes, OsNPR1/NH1, OsNPR2/NH2 and OsNPR3, were found to be induced by rice bacterial blight Xanthomonas oryzae pv. oryzae and rice blast Magnaporthe grisea, and the defence molecules benzothiadiazole, methyl jasmonate and ethylene. We confirmed that OsNPR1 is the rice orthologue by complementing the Arabidopsis npr1 mutant. Over-expression of OsNPR1 conferred disease resistance to bacterial blight, but also enhanced herbivore susceptibility in transgenic plants. The OsNPR1-green fluorescent protein (GFP) fusion protein was localized in the cytoplasm and moved into the nucleus after redox change. Mutations in its conserved cysteine residues led to the constitutive localization of OsNPR1(2CA)-GFP in the nucleus and also abolished herbivore hypersensitivity in transgenic rice. Different subcellular localizations of OsNPR1 antagonistically regulated SA- and jasmonic acid (JA)-responsive genes, but not SA and JA levels, indicating that OsNPR1 might mediate antagonistic cross-talk between the SA- and JA-dependent pathways in rice. This study demonstrates that rice has evolved an SA-mediated systemic acquired resistance similar to that in Arabidopsis, and also provides a practical approach for the improvement of disease resistance without the penalty of decreased herbivore resistance in rice.     

LMM5.1 and LMM5.4,two eukaryotic translation elongation factor 1Alike gene family members,negatively affect cell death and disease resistance in rice

Lesion mimic mutant(LMM) genes, stimulating lesion formation in the absence of pathogens, play significant roles in immune response. In this study, we characterized a rice lesion mimic mutant, lmm5,which displayed light-dependent spontaneous lesions. Additionally, lmm5 plants exhibited enhanced resistance to all of the tested races of Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae(Xoo) by increasing the expression of defense-related genes and the accumulation of hydrogen peroxide. Genetic analysis showed that the lesion mimic phenotype of lmm5 was controlled by two genes, lmm5.1 and lmm5.4, which were isolated with a map-based cloning strategy. Remarkably, LMM5.1 and LMM5.4 share a 97.4% amino acid sequence identity, and they each encode a eukaryotic translation elongation factor 1A(e EF1A)-like protein. Besides, LMM5.1 and LMM5.4 were expressed in a tissue-specific and an indicaspecific manner, respectively. In addition, high-throughput m RNA sequencing analysis confirmed that the basal immunity was constitutively activated in the lmm5 mutant. Taken together, these results suggest that the homologous e EF1A-like genes, LMM5.1 and LMM5.4, negatively affect cell death and disease resistance in rice.     

Rice Pti1a negatively regulates RAR1-dependent defense responses

Tomato (Solanum lycopersicum) Pto encodes a protein kinase that confers resistance to bacterial speck disease. A second protein kinase, Pti1, physically interacts with Pto and is involved in Pto-mediated defense signaling. Pti1-related sequences are highly conserved among diverse plant species, including rice (Oryza sativa), but their functions are largely unknown. Here, we report the identification of a null mutant for the Pti1 homolog in rice and the functional characterization of Os Pti1a. The rice pti1a mutant was characterized by spontaneous necrotic lesions on leaves, which was accompanied by a series of defense responses and resistance against a compatible race of Magnaporthe grisea. Overexpression of Pti1a in rice reduced resistance against an incompatible race of the fungus recognized by a resistance (R) protein, Pish. Plants overexpressing Pti1a were also more susceptible to a compatible race of the bacterial pathogen Xanthomonas oryzae pv oryzae. These results suggest that Os Pti1a negatively regulates defense signaling for both R gene-mediated and basal resistance. We also demonstrated that repression of the rice RAR1 gene suppressed defense responses induced in the pti1a mutant, indicating that Pti1a negatively regulates RAR1-dependent defense responses. Expression of a tomato Pti1 cDNA in the rice pti1a mutant suppressed the mutant phenotypes. This contrasts strikingly with the previous finding that Sl Pti1 enhances Pto-mediated hypersensitive response (HR) induction when expressed in tobacco (Nicotiana tabacum), suggesting that the molecular switch controlling HR downstream of pathogen recognition has evolved differently in rice and tomato.     

Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice

New evidence suggests a role for the plant growth hormone auxin in pathogenesis and disease resistance. Bacterial infection induces the accumulation of indole-3-acetic acid (IAA), the major type of auxin, in rice (Oryza sativa). IAA induces the expression of expansins, proteins that loosen the cell wall. Loosening the cell wall is key for plant growth but may also make the plant vulnerable to biotic intruders. Here, we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid signaling- and jasmonic acid signaling-independent pathway. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation. Overexpression of GH3-8 results in enhanced disease resistance to the rice pathogen Xanthomonas oryzae pv oryzae. This resistance is independent of jasmonic acid and salicylic acid signaling. Overexpression of GH3-8 also causes abnormal plant morphology and retarded growth and development. Both enhanced resistance and abnormal development may be caused by inhibition of the expression of expansins via suppressed auxin signaling.     

Pseudomonas aeruginosa inducing rice resistance againstRhizoctonia solani: Production of salicylic acid and peroxidases

Three isolates of Pseudomonas aeruginosa were used for seed treatment of rice; all showed plant growth promoting activity and induced systemic resistance in rice against Rhizoctonia solani G5 and increased seed yield. Production of salicylic acid (Sal) by P. aeruginosa both in vitro and in vivo was quantified with high performance liquid chromatography. All three isolates produced more Sal in King's B broth than in induced roots. Using a split root system, more Sal accumulated in root tissues of bacterized site than in distant roots on the opposite site of the root system after 1 d, but this difference decreased after 3 d. Sal concentration 0-200 g/L showed no inhibition of mycelial growth of R. solani in vitro, while at > or =300 g/L it inhibited it. P. aeruginosa-pretreated rice plants challenged inoculation with R. solani (as pathogen), an additional increase in the accumulation of peroxidase was observed. Three pathogenesis-related peroxidases in induced rice plants were detected; molar mass of these purified peroxidases was 28, 36 and 47 kDa. Purified peroxidase showed antifungal activity against phytopathogenic fungi R. solani, Pyricularia oryzae and Helminthosporium oryzae.     

非寄主抗病基因Rxo1介导的水稻对Xanthomonas oryzae pv.oryzicola的抗病反应

通过表型鉴定、反转录PCR和实时定量PCR方法,利用转基因和非转基因水稻植株,研究由Rxol基因介导的,水稻对细菌性条斑病菌的抗性反应。结果观察到3个涉及过敏性反应的基因由Rxol基因诱导表达,并对其进行了分析。这3个基因参与编码病程相关蛋白,在转基因水稻植株中呈上调表达,表明水杨酸信号转导途径在抗性反应中发挥重要的作用。     

Overexpression of (At)NPR1 in rice leads to a BTH- and environment-induced lesion-mimic/cell death phenotype

Systemic acquired resistance (SAR) is an inducible defense response that protects plants against a broad spectrum of pathogens. A central regulator of SAR in Arabidopsis is NPR1 (nonexpresser of pathogenesis-related genes). In rice, overexpression of Arabidopsis NPR1 enhances plant resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. This report demonstrates that overexpression of (At)NPR1 in rice also triggers a lesion-mimic/cell death (LMD) phenotype. The LMD phenotype is environmentally regulated and heritable. In addition, the development of lesions and death correlates with the expression of rice defense genes and the accumulation of hydrogen peroxide. Application of the salicylic acid (SA) analog, benzo(1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH), potentiates this phenotype Endogenous SA levels are reduced in rice overexpressing (At)NPR1 when compared with wildtype plants, supporting the idea that (At)NPR1 may perceive and modulate the accumulation of SA. The association of (At)NPR1 expression in rice with the development of an LMD phenotype suggests that (At)NPR1 has multiple roles in plant stress responses that may affect its efficacy as a transgenic tool for engineering broad-spectrum resistance.     

Molecular characterization of a pathogenesis-related protein 8 gene encoding a class III chitinase in rice

A cDNA encoding a class III chitinase (Oschib1) was isolated from a cDNA library constructed from rice leaves infected with the blast fungus Magnaporthe grisea. The cDNA contains an open reading frame of 861 nucleotides encoding 286 amino acid residues with a pI of 5.06. The deduced amino acid sequence of Oschibl has a high level of similarity with class IIIb chitinases of Gladiolus gandavensis (46%) and Tulipa bakeri (49%). A high level of Oschibl mRNA was detected after inoculation with M. grisea or Xanthomonas oryzae pv. oryzae. Expression of Oschib1 was induced more rapidly when an avirulent strain of M. grisea was inoculated (incompatible interaction) than when a virulent strain was used (compatible interaction). Expression of Oschibl was also induced by treatment of signaling molecules such as salicylic acid, ethylene, and methyl jasmonic acid, and by treatment with H2O2 or CuSO4. The induction patterns of Oschibl expression suggest that Oschib1 may be involved in defense response against pathogen infections and may be classified as a member of pathogenesis-related protein 8 in rice.     

A MAP kinase gene, BMK1, is required for conidiation and pathogenicity in the rice leaf spot pathogen Bipolaris oryzae

We isolated and characterized BMK1, a gene encoding a mitogen-activated protein kinase (MAPK), from the rice leaf spot pathogen Bipolaris oryzae. The deduced amino acid sequence showed significant homology with Fus3/Kss1 MAPK homologues from other phytopathogenic fungi. The BMK1 disruptants showed impaired hyphal growth, no conidial production, and loss of virulence against rice leaves, indicating that the BMK1 is essential for conidiation and pathogenicity in B. oryzae.     

RMo1 confers blast resistance in barley and is located within the complex of resistance genes containing Mla, a powdery mildew resistance gene

Isolates of Magnaporthe oryzae (the causal agent of rice blast disease) can infect a range of grass species, including barley. We report that barley Hordeum vulgare cv. Baronesse and an experimental line, BCD47, show a range of resistance reactions to infection with two rice blast isolates. The complete resistance of Baronesse to the isolate Ken 54-20 is controlled by a single dominant gene, designated RMo1. RMo1 mapped to the same linkage map position on chromosome 1H as the powdery mildew resistance locus Mla and an expressed sequence tag (k04320) that corresponds to the barley gene 711N16.16. A resistance quantitative trait locus (QTL), at which Baronesse contributed the resistance allele, to the isolate Ken 53-33 also mapped at the same position as RMo1. Synteny analysis revealed that a corresponding region on rice chromosome 5 includes the bacterial blight resistance gene xa5. These results indicate that a defined region on the short arm of barley chromosome 1H, including RMo1 and Mla, harbors genes conferring qualitative and quantitative resistance to multiple pathogens. The partial resistance of BCD47 to Ken53-33 is determined by alleles at three QTL, two of which coincide with the linkage map positions of the mildew resistance genes mlo and Mlf.     

PhyA, a secreted protein of Xanthomonas oryzae pv. oryzae, is required for optimum virulence and growth on phytic acid as a sole phosphate source

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. We have identified a novel virulence deficient mutant (BXO1691) of X. oryzae pv. oryzae that has a Tn5 insertion in an open reading frame (phyA; putative phytase A) encoding a 373-amino acid (aa) protein containing a 28-aa predicted signal peptide. Extracellular protein profiles revealed that a 38-kDa band is absent in phyA mutants as compared with phyA+ strains. A BLAST search with phyA and its deduced polypeptide sequence indicated significant similarity with conserved hypothetical proteins in Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris and limited homology to secreted phytases of Bacillus species. Homology modeling with a Bacillus phytase as the template suggests that the PhyA protein has a similar six-bladed beta-propeller architecture and exhibits conservation of certain critical active site residues. Phytases are enzymes that are involved in degradation of phytic acid (inositol hexaphosphate), a stored form of phosphate in plants. The phyA mutants exhibit a growth deficiency in media containing phytic acid as a sole phosphate source. Exogenous phosphate supplementation promotes migration of phyA X. oryzae pv. oryzae mutants in rice leaves. These results suggest that the virulence deficiency of phyA mutants is, at least in part, due to inability to use host phytic acid as a source of phosphate. phyA-like genes have not been previously reported to be involved in the virulence of any plant pathogenic bacterium.     

Functional interplay between two Xanthomonas oryzae pv,. oryzae secretion systems in modulating virulence on rice

The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S-) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S- T3S- double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA- mutant or a ClsA- LipA- double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.     

The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated in stability control of rice receptor-like kinase

The rice gene Xa21 confers resistance against Xanthomonas oryzae pv. oryzae (Xoo). Xa21 encodes a receptor-like kinase (XA21). We demonstrate that XA21 autophosphorylates residues Ser686, Thr688 and Ser689 in vitro. Substitution of these residues with alanines did not affect the autophosphorylation function of this kinase, but specifically destabilized the resistance protein in vitro and in vivo. Plants carrying these same substitutions in XA21 were compromised in their resistance to the normally avirulent Xoo Philippine race 6. Additionally, we show that wild-type XA21 and the kinase-dead mutant with the invariable Lys736 residue mutated to glutamic acid were also proteolytically degraded in protein extracts. Finally, we show a correlation between the in vitro degradation and in vivo instability of the proteins. We propose that autophosphorylation of Ser686, Thr688 and Ser689 functions to stabilize XA21 against the developmentally controlled proteolytic activity.     

Ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae

The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99(A) and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice.     

A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice (Oryza sativa L.) blast resistance allele of Pid3, designated Pi25, from a stable blast resistance cultivar Gumei2. Overexpression analysis and complementation test showed that Pi25 conferred blast resistance to M. oryzae isolate js001-20. Sequence analysis showed that Pi25 was an intronless gene of 2772 nucleotides with single nucleotide substitution in comparison to Pid3 at the nucleotide position 459 and predicatively encoded a typical coiled coil-nucleotide binding site-leucine rich repeat (CC--NBS--LRR) protein of 924 amino acid residuals with 100% identity to Pid3 putative protein. The susceptible allele pi25 in Nipponbare contained a nonsense mutation at the nucleotide position 2209 resulting in a truncated protein with 736 amino acid residuals. In addition, 14 nucleotide substitutions resulting in 10 amino acid substitutions were identified between Pi25 and pi25 upstream the premature stop codon in the susceptible allele. Although the mechanism of Pi25/Pid3-mediated resistance needs to be further investigated, the isolation of the allele would facilitate the utilization of Pi25/Pid3 in rice blast resistance breeding program via transgenic approach and marker assisted selection.