Molecular Screening of Rice Cultivated in Benin for the Identification of Xanthomonas oryzae Pv. oryzae and Bacterial Leaf Blight Resistance Genes

One of the most devastating diseases of rice worldwide is bacterial blight (BLB) caused by Xanthomonas oryzae pv. Oryzae (Xoo). In Benin, Xoo was first described in 2013 on wild rice Oryzae longistaminata. So far, no study has been done on Beninese Xoo strains. We do not know whether the pathogen has already passed into the rice varieties grown, or if they are exposed to other bacteria. Whereas the use of resistant varieties, carrying resistance genes, is the only highly effective and environmentally friendly way to control this disease, no information is available on these Xoo resistance genes in rice varieties grown in Benin apart from the one we recently. This study aims to identify Beninese Xoo strains, causing BLB and screen rice varieties grown in Benin for the main resistance genes. Diseased rice leaves showing typical symptoms of fire blight collected from different rice fields in the three phytogeographic areas of Benin were analyzed by PCR for Xoo-specific sequence identification. Furthermore, seventy-five collected rice accessions were screened to identify xa5, Xa7, xa13, and Xa21 resistance genes to Xoo. The results reveal that Xanthomonas oryzae was identified in two fields in Banikouara and one in Malanville. On the other hand, Sphingomonas sp. has been identified in several other rice fields in Benin. Forty-seven of seventy-five rice accessions examined (62.66%) carried Xoo resistance genes with 3 (4%) and 40 (53.33%) of xa5 and Xa21 respectively. None of the accessions had either Xa7 or xa13 resistance genes. Three accessions possess both xa5 and Xa21 genes. Isogenic lines IRBB60 and IRBB21, supposed to be a positive control, presented a Xoo sensitivity allele. These results indicate that Xoo has moved from the wild rice variety to the cultivated variety in northern Benin and varietal improvement programs must be implemented with varieties having several resistance genes for the efficient response against a possible BLB pandemic in Benin.

Establishment of a system for screening and identification of novel bactericide targets in the plant pathogenic bacterium Xanthomonas oryzae pv. oryzae using Tn-seq and SPR

Xanthomonas spp. cause severe bacterial diseases. However, effective strategies for prevention and management of these diseases are scarce. Thus, it is necessary to improve the efficiency of control of diseases caused by Xanthomonas. In this study, Xanthomonas oryzae pv. oryzae(Xoo), which causes rice bacterial leaf blight, has been studied as a representative. A transposon insertion library of Xoo, comprising approximately 200,000 individual insertion mutants, was generated. Transposon sequencing data indicated that the mariner C9 transposase mapped at 35.7–36.4% of all potential insertion sites, revealing 491 essential genes required for the growth of Xoo in rich media. The results show that, compared to the functions of essential genes of other bacteria, the functions of some essential genes of Xoo are unknown, 25 genes might be dangerous for the Xanthomonas group, and 3 are specific to Xanthomonas. High-priority candidates for developing broad-spectrum, Xanthomonas-specific, and environment-friendly bactericides were identified in this study. In addition, this study revealed the possible targets of dioctyldiethylenetriamine using surface plasmon resonance(SPR) in combination with high performance liquid chromatography–mass spectrometry(HPLC–MS). The study also provided references for the research of some certain bactericides with unknown anti-bacterial mode of action. In conclusion, this study urged a better understanding of Xanthomonas,provided meaningful data for the management of bacterial leaf blight, and disclosed selected targets of a novel bactericide.

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

An improved protein expression system for T3SS genes regulation analysis in Xanthomonas oryzae pv. oryzae

Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

Functional identification of phenazine biosynthesis genes in plant pathogenic bacteria Pseudomonas syringae pv. tomato and Xanthomonas oryzae pv. oryzae

Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identified phenazine biosynthesis （phz） genes in two genome-completed plant pathogenic bacteria Pseudomonas syringae pv. tomato （Pst） DC3000 and Xanthomonas oryzae pv. oryzae （Xoo） PXO99A. Unlike the phz genes in typical phenazine-producing pseudomonads, phz homologs in Pst DC3000 and Xoo PXO99A consisted of phzC/D/E/F/G and phzC/E1/E2/F/G, respectively, and the both were not organized into an operon. Detection experiments demonstrated that phenazine-l-carboxylic acid （PCA） of Pst DC3000 accumulated to 13.4 IJg L-1, while that of Xoo PXO99A was almost undetectable. Moreover, Pst DC3000 was resistant to 1 mg mL-1 PCA, while Xoo PXO99A was sensitive to 50 IJg mL ~ PCA. Furthermore, mutation of phzF blocked the PCA production and significantly reduced the pathogenicity of Pst DC3000 in tomato, while the complementary strains restored these phenotypes. These results revealed that Pst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologically controlled by phenazines. Additionally, phz-mediated PCA production is required for full pathogenicity of Pst DC3000. To our knowledge, this is the first report of PCA production and its function in pathogenicity of a plant pathogenic P. syringae strain.

Virulence of Xanthomonas oryzae pv. oryzae on Rice Near-lsogenic Lines with Single Resistance Gene and＇Pyramiding Lines in China

Ninety one isolates of Xanthomonas oryzae pv. oryzae were collected from different rice- growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene and 2-4 genes: IRBB1 (Xa1), IRBB2 (Xa2), IRBB3 (Xa3), IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11), IRBB13 (xa13), IRBB14 (Xa14), IRBB21 (Xa21), IR24 (Xa18), IRBB50 (Xa4 + xa5), IRBB51 (Xa4 + xa13), IRBB52 (Xa4 + Xa21), IRBB53 (xa5 + xa13), IRBB54 (xa5 + Xa21), IRBB55 (xa13 + Xa21), IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21), IRBB59 (xa5 + xa13 + Xa21) and IRBB60 (Xa4 + xa5 + xa13 + Xa21). The results showed that most isolates were less virulent on lines with more than one genes pyramided than those with single resistance gene. The isolates tested were more virulent on IR24 and IRBB10, less virulent on IRBB5, IRBB7 and IRBB21. Based on interactions between isolates and rice near-isogenic lines, 7 cultivars with single gene (IRBB5, IRBB4, IRBB3, IRBB14, IRBB2, IRBB1 and IR24) were chosen as the differentials, and the tested isolates were classified into 7 virulence groups. The reaction patterns of the 7 groups in order were: RRRRRRR, RRRRRRS, RRRRRSS, RR/SRRSSS, RRRSSSS, RRSSSSS, RSSSSSS. The virulence frequencies were 7.69, 6.59, 14.29, 12.09, 14.29, 28.57 and 16.48% respectively. The elementary system for races identification has been established in China based on the results. It will be possible to compare with races in other countries, and the results will facilitate the development of rice resistance breeding to bacterial blight in China.

Exchangeability of Two hrp Gene Fragments from Xanthomonas oryzae pv.oryzae and pv.oryzicola for Hypersensitive Response on Tobacco and Pathogenicity on Rice

hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che mical. All the hrp mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell sonicated integrations of the wild type strains and the hrp mutants demonstrated that there existed an HR eliciting substance which was heat stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6 kb hrp fragment insert in pUHRX245 and a 39.3 kb insert in pUHRS138 revealed that a 3.3 kb Sac Ⅰ fragment from pUHRX245 and a 4.5 kb Bam HⅠ Kpn Ⅰ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3 kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.

An Efficient Electro-Competent Cells Generation Method of Xanthomonas campestris pv. campestris: Its Application for Plasmid Transformation and Gene Replacement

A simple and rapid method to prepare efficient electro-competent cells of Xanthomonas campestris pv. campestris was generated, with up to 100-fold transformation efficiencies over the existing procedures. The overnight cultures were treated with sucrose solution and micro-centrifuged at room temperature;the entire electro-competent cells generation process can be completed in 15 minutes. It overcomes the complication and time-consuming shortcomings of the traditional conjugation or electro-transformation methods in this strain. Both the replicative plasmids and non-replicative plasmids could be transformed or integrated efficiently using this method. And the DNA concentration, cells growth stage, field strength and recovery time all had influences on the transformation efficiency. In the optimal conditions, the transformation efficiency for the replicative plasmids was 109 transformants per microgram DNA, and for non-replicative plasmids was 150 transformants per microgram DNA. Further with the homology sequences, two chromosomal target genes were deleted efficiently and the knockout strains were obtained easily.

Evolution of Xanthomonas Gene Content:Gene Gain/Loss History and Species Divergence

Horizontal gene transfer （HGT） plays key roles in the evolution of pathogenetic bacteria, especially in pathogenetic associated genes. In this study, the evolutionary dynamics of Xanthomonas at species level were determined by the comparative analysis of the complete genomes of 15 Xanthomonas strains. A concatenated multiprotein phyletic pattern and a dataset with Xanthomonas clusters of orthologous genes were constructed. Mathematical extrapolation estimates that the core genome will reach a minimum of about 1 547 genes while the pan-genome will increase up to 22 624 genes when sequencing 1 000 genomes. The HGT extent in this genus was assessed by using a Markov-based probabilistic method. The reconstructed gene gain/loss history, which contained several features consistent with biological observations, showed that nearly 60% of the Xanthomonas genes were acquired by HGT. A large fraction of variability was in the clade ancestor nodes and ＂leaves of the tree＂. Coexpression analysis suggested that the pathogenic and metabolic variation between Xanthomonas oryzae pv. oryzicola and Xanthomonas oryzae pv. oryzae might due to recently-transferred genes. Our results strongly supported that the gene gain/loss may play an important role in divergence and pathogenicity variation of Xanthomonas species.

野油菜黄单胞菌(Xanthomonas campestris)抗药性质粒转化研究

本论文研究了野油菜黄单胞菌(Xanthomonascampestris)菌种的不同生长状态、不同转化液等因素对感受态细胞转化率的影响。旨在通过各方面因素的综合比较,获得一个最佳的菌种抗药转化方案。

黄单胞菌(Xanthomonas campestris)XA5-5β-葡萄糖苷酶基因的克隆与表达

以黄单胞菌(Xanthomonas campestris)XA5-5为供体菌,广泛寄主质粒pRK404为载体,在大肠杆菌中克隆了一个β-葡萄糖苷酶基因,重组质粒pLZS1外源片段为1.1kb,将pLZS1接合导入黄单胞菌XA5-5,得到了克隆子XA5-5(pLZS1).以水杨苷为底物,XA5-5(pLZS1)的酶活性大大高于E.Coli JM83(pLZS1).并且质粒在XA5-5中能相对稳定地存在.初步结果表明,所克隆的基因编码的产物对于水杨苷底物有较强的亲和力,并可以在一定程度上降低XA5-5中酶与pNPG底物的亲和力,使其酶活减小.

The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp.citri

Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citrisubsp. citri, carA and carB encode the small and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hyper- sensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type Ill secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracellular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp, citri.

Diversity and interaction of common bacterial blight disease-causing bacteria(Xanthomonas spp.) with Phaseolus vulgaris L.

Common bacterial blight(CBB) is associated with common bean(Phaseolus vulgaris L.), an important grain legume for human consumption worldwide. The disease, caused by Xanthomonas spp. is spread mainly through seed. This paper focuses on the diversity of X.axonopodis pv. phaseoli and X. fuscans subsp. fuscans and interactions between related bacteria and the bean host. Review has suggested that the diversity and taxonomic studies of these pathogens are not exhaustive, especially in areas where detailed molecular analysis has not been conducted and previous characterizations were based on phenotypic features and PCR-based techniques. Also, no study has confirmed differential pathogenicity on bean genotypes based on compatible versus incompatible reactions. However, isolates react differently to wild and domesticated bean sources of resistance in common bean genetic backgrounds. A systematic approach will be required to investigate global changes in gene expression among different sources of resistance in a common bean background.The bacterial isolates that cause CBB should be functionally characterized using genotypes containing major quantitative trait loci(QTL) for CBB resistance. These studies will increase understanding of resistance and how it is manipulated by pathogens.

水稻白叶枯病黄单胞菌中与甘蓝黑腐病黄单胞菌致病因子调控基因(rpf 基因)同源基因的分子克隆

DNA-DNA 杂交结果表明,水稻白叶枯病黄单胞菌总 DNA 中存在着与甘蓝黑腐病黄单胞菌致病因子调控基因(rpf 基因)具有同源性的 DNA 外段.应用广谱性寄主载体pLAFRI,在大肠杆菌中建立了水稻白叶枯病黄单胞菌野生型菌株 T3000的基因文库.从文库中筛选到与 rpf 基因具有同源性的克隆 pGXN3000,将重组质粒 pGXN3000通过三亲本接合转入甘蓝黑腐病黄单胞菌 rpf 基因突变体,发现 pGXN3000能互补甘蓝黑腐病黄单胞菌 rpf突变体,恢复其致病性胞外酶和胞外多糖的产生及致病性.转座子 Tn5诱变,缺失分析及DNA-DNA 杂交实验结果表明,重组质粒 pGXN3000中确实含有与甘蓝黑腐病黄单胞菌编码。双组份调控系统”蛋白的 rpfC 基因功能相同的基因,此基因被定位于 pGXN3000中的3kb BamHl 片段中。

水稻黄单胞菌水稻变种的rpfA基因的定位和次级克隆

水稻黄单胞菌水稻变种产生两种顺乌头酸酶（ＸｏｏＡｃａｎＡ和ＸｏｏＡｃａｎＢ），编码ＸｏｏｃａｎＡ的ｒｐｆＡ基因已被克隆在重组质粒ｐＧＸＮ３０００中。本工作通过转座子Ｔｎ５Ｂ２０诱变ｐＧＸＮ３０００，鉴定了ｒｐｆＡ基因的位置及其转录方向，并进一步将含有完整ｒｐｆＡ基因的４．８ｋｂＡｓｐ７１８ＥｃｏＲⅠ片段次级克隆到了多用途载体质粒ｐＩＪ３２００。

水稻白叶枯病菌hrpF启动子序列调控的绿色荧光蛋白基因表达体系的构建

为了阐明启动子序列对水稻白叶枯病菌效应子基因表达的调控作用及特征,通过融合PCR将报告基因GFP基因置于水稻白叶枯病菌hrpF基因启动子序列的下游,经酶切、PCR鉴定及序列测定,成功构建了受hrpF启动子序列调控的GFP基因转录单元pMF-GFP,并转入JXO I菌株中。结果发现构建的hrpF::GFP转录单元,在hrp诱导培养基XOM2上可有效诱导表达,而在NA培养基上不能诱导表达。结果还显示,在大肠杆菌中,中间表达载体phrpF::GFP具有GFP表达活性。

云南高原粳稻白叶枯病菌的抗药性室内鉴定及其rpfC基因序列分析

为了探索云南省高原粳稻上十种不同致病型的白叶枯病菌对噻枯唑、叶枯灵和新植霉素三种农药的抗药性及其机制,分别用含有不同浓度农药的 NA 培养基进行白叶枯病菌的室内抗药性筛选,并设计与菌株抗药性密切相关的 rpfC 基因特异引物,对抗药性不同的菌株进行扩增、测序、基因和氨基酸序列比对分析.结果表明,噻枯唑对参试的所有菌株的最小抑菌浓度（MIC）为40~180 mg/L,而叶枯灵为10~100 mg/L,没有发现对新植霉素产生抗性的菌株；病原菌的致病型与对农药的敏感性相关,致病性强的菌株,其抗药性较强.将致病型和抗药性不同的10个菌株的 rpfC 基因序列与 GenBank 中登录号为 X97865．1的基因序列比对,序列同源性为92％~98％,而 RpfC 蛋白序列同源性差异较大（8．3％至99％）.致病性和抗药性最强的Ⅵ型菌株2001-31的 RpfC 蛋白序列的六个功能域完整,致病性和抗药性最弱的0型菌株 DH-L-1的 RpfC 蛋白序列的信号接收区域 REC 已经消失.7个致病性和抗药性中等的菌株中,Ⅳ型菌株⑤较为特殊,其 RpfC 蛋白序列已经不能形成功能域.

水稻黄单胞菌水稻致病变种rpfC缺失突变体在感病和抗病水稻品种中的繁殖和扩展

水稻黄单胞菌水稻致病变种（Ｘａｎｔｈｏｍｏｎａｓｏｒｙｚａｅｐｖ．ｏｒｙｚａｅ）中国菌株１３７５１和水稻品种广桂１１０和ＩＲ２６的相互作用分别为亲和和不亲和相互作用。１３７５１的ｒｐｆＣ基因缺失突变株ＳＬ３７５１在电镜下的形态和野生型没有显著差别，均为短杆状，大小为（０．５～０．８）×（１．０～２．０）μｍ。用１０８ｃｆｕ／ｍＬ或１０１０ｃｆｕ／ｍＬ的ＳＬ３７５１剪叶接种苗期和分蘖盛期的广桂１１０，接种后５ｄ内ＳＬ３７５１在广桂１１０中的生长繁殖和野生型相似，但接种５ｄ后ＳＬ３７５１在其中的活菌数均稳定在１０６－７ｃｆｕ／叶，最终ＳＬ３７５１的活菌数比１３７５１的低１００倍左右。在抗病品种ＩＲ２６上，接种后５ｄＳＬ３７５１也增殖到１０６－７ｃｆｕ／叶，之后也保持在这一水平，最终ＳＬ３７５１的活菌数比野生型的低１０倍左右。另外，ＳＬ３７５１在被接种到广桂１１０和ＩＲ２６后，它被主要局限在剪叶接种切口下３ｃｍ范围内。野生型接种在广桂１１０上，接种后３ｄ就迅速沿叶脉往下扩展，野生型１３７５１在抗病品种ＩＲ２６中的扩展速度显著慢于其在广桂１１０中的扩展速度。所有这些都进一步证实ｒｐｆＣ基因在１３７５１在致病过程中起?

水稻黄单胞杆菌水稻变种rpfA基因与毒性相关

水稻黄单胞杆菌水稻变种产生两种顺乌头酸酶 (XooAcanA和XooAcanB)。编码其中的XooAcanA的rpfA基因已被克隆在重组质粒 pGXN30 0 0中。本研究通过转座子诱变 pGXN30 0 0及标记置换方法 ,构建了rpfA突变体T3A0 1。虽然T3A0 1丧失了合成顺乌头酸梅XooAcanA的能力 ,但仍然能够合成顺乌头酸酶XooAcanB。植株实验结果表明 ,该突变体的生长速率与野生型相似 ,但是引起的症状与野生型相比明显减弱 ,说明水稻黄单胞杆菌水稻变种的rpfA基因与毒性相关。

Identification of a Resistance Gene bls1 to Bacterial Leaf Streak in Wild Rice Oryza rufipogon Griff

Bacterial leaf streak （BLS） of rice, caused by Xanthomonas oryzae pv. oryzicola （Xoc） is a worldwide destructive disease. Development of resistant varieties is considered to be one of the most effective and eco-friendly ways to control the disease. However, only a few genes/QTLs having resistance to BLS have been identified in rice until now. In the present study, we have identified and primarily mapped a BLS-resistance gene, blsl, from a rice line DP3, derived from the wild rice species Oryza rufipogon Griff. A BC2F2 （9311/DP3//9311） population was constructed to map BLS-resistance gene in the rice line DP3. The segregation of the resistant and susceptible plants in BCzFz in 1：3 ratio （Z2=0.009, Z20 05,1=3.84, P〉0.05）, suggested that a recessive gene confers BLS resistance in DP3. In bulked segregant analysis （BSA）, two SSR markers RM8116 and RM584 were identified to be polymorphic in resistant and susceptible DNA bulks. For further mapping the resistance gene, six polymorphic markers around the target region were applied to analyze the genotypes of the BC2F2 individuals. As a result, the BLS-resistant gene, designated as blsl, was mapped in a 4.0-cM region flanked by RM587 and RM510 on chromosome 6.