Xanthomonas oryzae pv. oryzae infection triggers accumulation of phenolics,defense-related enzymes and thaumatin-like proteins in rice leaves

Abstract

The effect of Xanthomonas oryzae pv. oryzae infection on induction of phenylalanine ammonia-lyase (PAL), peroxidase (PO), phenolics and thaumatin-like proteins (TLPs) in rice was studied. PAL activity increased significantly one day after inoculation with X. o. pv. oryzae and the maximum enzyme activity was observed two days after inoculation. The phenolic content in rice leaves increased significantly one day after inoculation and the maximum accumulation of phenols was observed two days after inoculation. Significant increase in peroxidase activity was observed in rice leaves one day after inoculation with X. o. pv. oryzae. Isozyme analysis indicated that three peroxidase isozymes (PO-1, PO-2 and PO-3) were induced after inoculation with X. o. pv. oryzae. Immunoblot analysis of protein extracts from control and pathogen inoculated rice plants revealed the induced accumulation of 16 and 24 kDa TLPs in rice leaves in response to X. o. pv. oryzae infection. TLP mRNA accumulation was induced strongly in rice leaves in response to infection by X. o. pv. oryzae.     

Virulence Analysis and Race Classification of Xanthomonas oryzae pv. oryzae in China

Two hundred and eighty‐five isolates of Xanthomonas oryzae pv. oryzae were randomly collected from 22 rice‐growing provinces in China. Ninety‐one representative isolates were chosen to assess the differential characteristics of 24 near‐isogenic rice lines containing a single resistance gene or two to four genes. Most isolates were avirulent on pyramided lines, except IRBB51, and hence, the pyramided lines cannot be used as differentials for the virulence analysis of X. oryzae pv. oryzae in China. The 13 rice lines with a single gene were used further to establish a system of races classification of X. oryzae pv. oryzae in China. IR24 and IRBB10 were susceptible to the isolates with several exceptions, whereas IRBB5, IRBB7 and IRBB21 were resistant. Based on the interactions between the isolates of X. oryzae pv. oryzae and the 13 near‐isogenic rice lines, six single‐gene rice cultivars (IRBB5, IRBB13, IRBB3, IRBB14, IRBB2 and IR24) were chosen as differentials, and the 285 tested isolates were classified into nine races. The reaction patterns of the nine races in order were: RRRRRR, RRRRRS, RRRRSS, RRRSSS, RRSSSS, RSRRRS, RSSRRS, RSSSSS and SSSSSS. The race frequencies were 10.18%, 10.53%, 4.91%, 10.18%, 24.21%, 5.96%, 11.23%, 22.46% and 0.35% respectively. The virulence of representative strains of eight Philippine races on 13 rice lines with a single gene was determined and compared with the Chinese races. The frequency distributions of X. oryzae pv. oryzae races were primarily described for the different regions in China.     

Aberrant vegetative and reproductive development by overexpression and lethality by silencing of OsHAP3E in rice

We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase. In the reproductive phase, dense panicle was developed, and occasionally successive generation of lateral rachises and formation of double flowers were observed. These phenotypes indicate association of OsHAP3E with determination of floral meristem identity. On the other hand, repression of OsHAP3E by RNAi or by overexpressing chimeric repressor fusion constructs brought about lethality to transformed cells, and almost no transformant was obtained. This suggests that the OsHAP3E function is essential for rice cells. Altogether, our loss-of-function and gain-of-function analyses suggest that OsHAP3E plays important pleiotropic roles in vegetative and reproductive development or basic cellular processes in rice.     

Identification,characterization and interaction of <Emphasis Type="Italic">HAP</Emphasis> family genes in rice

A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5 genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB288027 to AB288048 and BR000373 to BR000375.     

Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco

The β-glucosidase gene of maize (ZmGLU1) was suggested to hydrolyze cytokinin-conjugate and release free cytokinin during plant growth and development. A clone containing the upstream region of ZmGLU1 was isolated and sequenced from a maize genomic library. The full-length ZmGLU1 promoter and a series of its 5′ deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred into tobacco. The GUS activity of transgenic plants was assayed at various developmental stages. The results showed that ZmGLU1 promoter-driven GUS gene had the highest expression level in the roots and that the expression of GUS gene declined during seed maturation and down to the lowest level in mature seeds. The ZmGLU1 promoter-driven GUS expression increased during seed germination, reaching a peak on day 11. The results also showed that this promoter could be inhibited by 6-BA, trans-zeatin, and NAA, but was not affected by GA₃, ABA, SA, cold, salt, drought, and submergence treatments. The histochemical staining revealed that GUS activity was located in vigorous cell division zones with dominant staining associated with vascular tissues. Deletion analysis showed that the promoter contained a putative leaf-specific and stem-specific negative regulative element and two putative enhancers.     

Global functional analyses of rice promoters by genomics approaches

Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. Promoters that are subject to specific regulations are useful for manipulating foreign gene expression in plant cells, tissues, or organs with desirable patterns and under controlled conditions, and have been important for both basic research and applications in agriculture biotechnology. Recent advances in genomics technologies have greatly facilitated identification and study of promoters in a genome scale with high efficiency. Previously we have generated a large T-DNA tagged rice mutant library (TRIM), in which the T-DNA was designed with a gene/promoter trap system, by placing a promoter-less GUS gene next to the right border of T-DNA. GUS activity screens of this library offer in situ and in planta identifications and analyses of promoter activities in their native configurations in the rice genome. In the present study, we systematically performed GUS activity screens of the rice mutant library for genes/promoters constitutively, differentially, or specifically active in vegetative and reproductive tissues. More than 8,200 lines have been screened, and 11% and 22% of them displayed GUS staining in vegetative tissues and in flowers, respectively. Among the vegetative tissue active promoters, the ratio of leaf active versus root active is about 1.6. Interestingly, all the flower active promoters are anther active, but with varied activities in different flower tissues. To identify tissue specific ABA/stress up-regulated promoters, we compared microarray data of ABA/stress induced genes with those of tissue-specific expression determined by promoter trap GUS staining. Following this approach, we showed that the peroxidase 1 gene promoter was ABA up-regulated by 4 fold within 1 day of exposure to ABA and its expression is lateral root specific. We suggest that this be an easy bioinformatics approach in identifying tissue/cell type specific promoters that are up-regulated by hormones or other factors. Su-May Yu and Swee-Suak Ko equally contributed to this work.     

厚藤脱水素基因IpDHN启动子IpDHN-Pro的克隆和调控转录活性分析

为了解厚藤(Ipomoea pes-caprae)脱水素基因IpDHN (GenBank登录号:KX426069)启动子的转录活性和对非生物胁迫和植物激素ABA的响应,通过染色体步移法克隆了IpDHN的上游启动子序列IpDHN-Pro,长度为974 bp。构建IpDHN-Pro调控下GUS转基因载体,转化拟南芥(Arabidopsis thaliana)植株获得IpDHN-Pro::GUS转基因植株并进行GUS染色,验证IpDHN-Pro启动转录活性以及在氯化钠、甘露醇、ABA处理后拟南芥GUS基因表达变化。结果表明,扩增获得的IpDHN-Pro序列包含多个顺式作用元件,包括1个ABRE、3个Myb转录因子结合位点、富含TC的重复序列以及Skn-1基序等。转基因拟南芥GUS染色及qRT-PCR表明该序列可驱动GUS基因在拟南芥稳定表达,且表达受高盐、渗透压及ABA的诱导。这表明IpDHN-Pro是一个盐旱、ABA诱导的启动子序列,可应用于相关的植物抗逆遗传工程研究。     

Abscisic Acid Promotes Susceptibility to the Rice Leaf Blight Pathogen Xanthomonas oryzae pv oryzae by Suppressing Salicylic Acid-Mediated Defenses

The plant hormone abscisic acid (ABA) is involved in a wide variety of plant processes, including the initiation of stress-adaptive responses to various environmental cues. Recently, ABA also emerged as a central factor in the regulation and integration of plant immune responses, although little is known about the underlying mechanisms. Aiming to advance our understanding of ABA-modulated disease resistance, we have analyzed the impact, dynamics and interrelationship of ABA and the classic defense hormone salicylic acid (SA) during progression of rice infection by the leaf blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Consistent with ABA negatively regulating resistance to Xoo, we found that exogenously administered ABA renders rice hypersusceptible to infection, whereas chemical and genetic disruption of ABA biosynthesis and signaling, respectively, led to enhanced Xoo resistance. In addition, we found successful Xoo infection to be associated with extensive reprogramming of ABA biosynthesis and response genes, suggesting that ABA functions as a virulence factor for Xoo. Interestingly, several lines of evidence indicate that this immune-suppressive effect of ABA is due at least in part to suppression of SA-mediated defenses that normally serve to limit pathogen growth. Resistance induced by the ABA biosynthesis inhibitor fluridone, however, appears to operate in a SA-independent manner and is likely due to induction of non-specific physiological stress. Collectively, our findings favor a scenario whereby virulent Xoo hijacks the rice ABA machinery to cause disease and highlight the importance of ABA and its crosstalk with SA in shaping the outcome of rice-Xoo interactions.     

Transgenic expression of the rice Xa21 pattern‐recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern‐recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21‐mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar ‘Gonja manjaya’ (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.     

Involvement of Gluconeogenic Pathway in Virulence of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases of rice. A phosphoenolpyruvate synthase (ppsA)‐disrupted mutant OSPAM was generated by homologous suicide plasmid integration. The mutant was unable to grow in medium with pyruvate or C₄‐dicarboxylates as the sole carbon source, compared with the wild‐type, indicating a disruption in ppsA function. The mutant showed a reduction in virulence on rice but still induced a hypersensitive response in tobacco. When the mutant was complemented, the response was recovered to wild‐type. These results suggested that X. oryzae pv. oryzae possesses only PPSA route in gluconeogenesis, which is necessary for virulence.     

Enhanced disease resistance and hypersensitivity to BTH by introduction of an NH1/OsNPR1 paralog

Non‐expresser of pathogenesis‐related genes 1 (NPR1) is the master regulator of salicylic acid‐mediated systemic acquired resistance. Over‐expression of Arabidopsis NPR1 and rice NH1 (NPR1 homolog1)/OsNPR1 in rice results in enhanced resistance. While there are four rice NPR1 paralogs in the rice genome, none have been demonstrated to function in disease resistance. To study rice NPR1 paralog 3, we introduced constructs into rice and tested for effects on resistance to infection by Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. While over‐expression of NH3 using the maize ubiquitin‐1 promoter failed to enhance resistance, introduction of an extra copy of NH3 driven by its own promoter (nNT‐NH3) resulted in clear, enhanced resistance. Progeny analysis confirms that the enhanced resistance phenotype, measured by Xoo‐induced lesion length, is associated with the NH3 transgene. Bacterial growth curve analysis indicates that bacterial population levels are reduced 10‐fold in nNT‐NH3 lines compared to control rice lines. The transgenic plants exhibit higher sensitivity to benzothiadiazole (BTH) and 2,6‐dichloroisonicotinic acid (INA) treatment as measured by increased cell death. Expression analysis of pathogenesis‐related (PR) genes showed that nNT‐NH3 plants display greatly enhanced induction of PR genes only after treatment with BTH. Our study demonstrates an alternative method to employ a regulatory protein to enhance plant defence. This approach avoids using undesirable constitutive, high‐level expression and may prove to be more practical for engineering resistance.     

Tapetum-specific expression of theOsg6B promoter-β-glucuronidase gene in transgenic rice

The promoter of an anther tapetum-specific gene,Osg6B, was fused to a-glucuronidase (GUS) gene and introduced into rice byAgrobacterium-mediated gene transfer. Fluorometric and histochemical GUS assay showed that GUS was expressed exclusively within the tapetum of anthers from the uninucleate microspore stage (7 days before anthesis) to the tricellular pollen stage (3 days before anthesis). This is the first demonstration of an anther-specific promoter directing tapetum-specific expression in rice.Abbreviations GUS ßGlucuronidase