The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.     

水稻病程相关PR1家族蛋白质在叶片生长及与白叶枯病菌互作反应中的表达

病程相关(PR)蛋白质经常被用作抗病反应的分子标记。利用免疫印迹(WB)技术检测了7个PR1家族蛋白质在水稻(Oryza sativa)叶片生长及与白叶枯病菌互作反应过程中的表达,发现6个PR1家族蛋白质在叶片生长中有表达。检测PR1蛋白质在Xa21介导的抗白叶枯病过程中的表达,结果显示PR1#052、PR1#072、PR1#073和PR1#121四个蛋白质在抗病反应后期呈上调或诱导表达,PR1#071则表达下调。进一步比较它们在抗病、感病和对照(Mock)反应中的表达丰度,发现在抗病和感病反应中的变化幅度均明显大于对照反应,推测这些PR蛋白质在水稻-白叶枯病菌互作反应中发挥作用。另外,对PR1基因上游启动子区的cis元件进行了分析。该研究初步揭示了水稻PR1家族蛋白质的表达谱,为进一步了解PR1蛋白质的功能提供了线索。     

Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.     

Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1, whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection.     

Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.     

Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.     

A rice serine carboxypeptidase-like gene OsBISCPL1 is involved in regulation of defense responses against biotic and oxidative stress

Serine carboxypeptidase-like proteins (SCPLs) comprise a large family of protein hydrolyzing enzymes that play roles in multiple cellular processes. During the course of study aimed at elucidating the molecular basis of induced immunity in rice, a gene, OsBISCPL1, encoding a putative SCPL, was isolated and identified. OsBISCPL1 contains a conserved peptidase S10 domain, serine active site and a signal peptide at N-terminus. OsBISCPL1 is expressed ubiquitously in rice, including roots, stems, leaves and spikes. Expression of OsBISCPL1 in leaves was significantly up-regulated after treatments with benzothiadiazole, salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid, and also up-regulated in incompatible interactions between rice and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants with constitutive expression of OsBISCPL1 were generated and disease resistance assays indicated that the OsBISCPL1-overexpressing plants showed an enhanced disease resistance against Pseudomonas syringae pv. tomato and Alternaria brassicicola. Expression levels of defense-related genes, e.g. PR1, PR2, PR5 and PDF1.2, were constitutively up-regulated in transgenic plants as compared with those in wild-type plants. Furthermore, the OsBISCPL1-overexpressing plants also showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stress-related genes. The results suggest that the OsBISCPL1 may be involved in regulation of defense responses against pathogen infection and oxidative stress.     

Ten rice peroxidases redundantly respond to multiple stresses including infection with rice blast fungus

Class III plant peroxidases are believed to function in diverse physiological processes including disease resistance and wound response, but predicted low substrate specificities and the presence of 70 or more isoforms have made it difficult to define a specific physiological function(s) for each gene. To select pathogen-responsive POX genes, we analyzed the expression profiles of 22 rice POX genes after infection with rice blast fungus. The expression of 10 POX genes among the 22 genes was induced after fungal inoculation in both compatible and incompatible hosts. Seven of the 10 POX genes were expressed at higher levels in the incompatible host than in the compatible host 6-24 h after inoculation by which time no fungus-induced lesions have appeared. Organ-specific expression and stress-induced expression by wounding and treatment with probenazole, an agrichemical against blast fungus, jasmonic acid, salicylic acid and 1-aminocyclopropane-1-carboxylate, a precursor of ethylene, indicated that rice POXs have individual characteristics and can be classified into several types. A comparison of the amino acid sequences of POXs showed that multiple isoforms with a high sequence similarity respond to stress in different or similar ways. Such redundant responses of POX genes may guarantee POX activities that are necessary for self-defense in plant tissues against environmental stresses including pathogen infection.     

Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp

The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifHi::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1-carboxylate oxidase synthase, peroxidase precursor, PR10-a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important.     

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.     

水稻中大麦Mlo和玉米Hm1抗病基因同源序列的分析和定位

大麦抗病基因Mlo和玉米抗病基因Hm1编码的产物不具有绝大多数植物抗病基因产物所含有的保守结构域。这两个抗病基因的作用机理也不符合基因对基因学说。从水稻中分离克隆了Mlo基因的同源序列OsMlo-1和玉米Hm1基因的同源序列DFR－1。利用水稻分子标记遗传连锁图,将OsMlo-1定位于水稻第六染色体的两俱RZ667和RG424之间;Osmlo-1距离这两个分子标记分别为20．6和6．0cM(centi-Morgan)。将DFR－1定位于水稻第一染色体两个分子标记R2635和RG462之间;DFR－1距离这两个分子标记分别为11．3和23．9cM。参照已发表的水稻分子标记连锁图,发现OsMlo-1和DFR－1的染色体位点分别与两个报道的水稻抗稻瘟病数量性状位点（QTL）有较好的对应关系。结果提示,水稻中与大麦Mlo 和玉米Hml同源的基因可能也参于抗病反应的调控。     

Jasmonic acid and salicylic acid activate a common defense system in rice

Jasmonic acid (JA) and salicylic acid (SA) play important roles in plant defense systems. JA and SA signaling pathways interact antagonistically in dicotyledonous plants, but, the status of crosstalk between JA and SA signaling is unknown in monocots. Our rice microarray analysis showed that more than half of the genes upregulated by the SA analog BTH are also upregulated by JA, suggesting that a major portion of the SA-upregulated genes are regulated by JA-dependent signaling in rice. A common defense system that is activated by both JA and SA is thus proposed which plays an important role in pathogen defense responses in rice.     

Overexpression of a rice defense-related F-box protein gene OsDRF1 in tobacco improves disease resistance through potentiation of defense gene expression

F-box proteins play important roles in plant growth/development and responses to environmental stimuli through targeting substrates into degradation machinery. A rice defense-related F-box protein gene, OsDRF1, was cloned and identified during a course of study aimed at elucidating the molecular basis of induced immunity in rice. OsDRF1 encodes a protein of 328 amino acids, containing a highly conserved F-box domain. Expression of OsDRF1 was induced upon treatment with benzothiadiazole (BTH), a chemical inducer of defense responses in rice. Moreover, in BTH-treated rice seedlings, expression of OsDRF1 was further induced by infection with Magnaporthe grisea, the rice blast fungus, compared with those in water-treated seedlings. OsDRF1 was also upregulated in rice seedlings after treatment with ABA. Overexpression of OsDRF1 in transgenic tobacco resulted in enhanced disease resistance against tomato mosaic virus (ToMV) and Pseudomonas syringae pv. tabaci and strengthened expression of defense-related genes after salicylic acid treatment or ToMV infection. Root elongation of the OsDRF1-overexpressing transgenic seedlings was significantly inhibited by ABA, indicating that overexpression of OsDRF1 resulted in increased ABA sensitivity. The results suggest that OsDRF1 plays a role in disease resistance via upregulating defense-related gene expression and that OsDRF1 may also be involved in the response to ABA.     

Benzothiadiazole-induced priming for potentiated responses to pathogen infection, wounding, and infiltration of water into leaves requires the NPR1/NIM1 gene in Arabidopsis

Systemic acquired resistance (SAR) is a plant defense state that is induced, for example, after previous pathogen infection or by chemicals that mimic natural signaling compounds. SAR is associated with the ability to induce cellular defense responses more rapidly and to a greater degree than in noninduced plants, a process called "priming." Arabidopsis plants were treated with the synthetic SAR inducer benzothiadiazole (BTH) before stimulating two prominent cellular defense responses, namely Phe AMMONIA-LYASE (PAL) gene activation and callose deposition. Although BTH itself was essentially inactive at the immediate induction of these two responses, the pretreatment with BTH greatly augmented the subsequent PAL gene expression induced by Pseudomonas syringae pv. tomato infection, wounding, or infiltrating the leaves with water. The BTH pretreatment also enhanced the production of callose, which was induced by wounding or infiltrating the leaves with water. It is interesting that the potentiation by BTH pretreatment of PAL gene activation and callose deposition was not seen in the Arabidopsis nonexpresser of PR genes 1/noninducible immunity 1 mutant, which is compromised in SAR. In a converse manner, augmented PAL gene activation and enhanced callose biosynthesis were found, without BTH pretreatment, in the Arabidopsis constitutive expresser of pathogenesis-related genes (cpr)1 and constitutive expresser of pathogenesis-related genes 5 mutants, in which SAR is constitutive. Moreover, priming for potentiated defense gene activation was also found in pathogen-induced SAR. In sum, the results suggest that priming is an important cellular mechanism in acquired disease resistance of plants that requires the nonexpresser of PR genes 1/noninducible immunity 1 gene.     

Disease resistance against Magnaporthe grisea is enhanced in transgenic rice with suppression of omega-3 fatty acid desaturases

Linolenic acid (18:3) is the most abundant fatty acid in plant membrane lipids and is a source for various oxidized metabolites, called oxylipins. 18:3 and oxylipins play important roles in the induction of defense responses to pathogen infection and wound stress in Arabidopsis. However, in rice, endogenous roles for 18:3 and oxylipins in disease resistance have not been confirmed. We generated 18:3-deficient transgenic rice plants (F78Ri) with co-suppression of two omega-3 fatty acid desaturases, OsFAD7 and OsFAD8. that synthesize 18:3. The F78Ri plants showed enhanced resistance to the phytopathogenic fungus Magnaporthe grisea. A typical 18:3-derived oxylipin, jasmonic acid (JA), acts as a signaling molecule in defense responses to fungal infection in Arabidopsis. However, in F78Ri plants, the expression of JA-responsive pathogenesis-related genes, PBZ1 and PR1b, was induced after inoculation with M. grisea, although the JA-mediated wound response was suppressed. Furthermore, the application of JA methyl ester had no significant effect on the enhanced resistance in F78Ri plants. Taken together, our results indicate that, although suppression of fatty acid desaturases involves the concerted action of varied oxylipins via diverse metabolic pathways, 18:3 or 18:3-derived oxylipins, except for JA, may contribute to signaling on defense responses of rice to M. grisea infection.