Effect of mechanical threshing on damage and vigor of maize seed threshed at different moisture contents

Mechanical threshing used when preparing maize seeds for planting subjects seed to damage and reduces seed quality.The purpose of this study was to assess the effect of mechanical threshing on the quality of maize seed threshed at different moisture contents(MCs).Seeds of dent maize JK968 and flint maize DD2 were threshed at 12,15,18,21,and 24% MC.The damage degree was determined by iodine staining,and seed vigor was assessed by standard germination(SG),cold test germination(CTG),accelerated aging germination(AAG),seedling emergence rate,and seedling root and shoot length.The results showed that the damage percentage increased,and the seed vigor parameters decreased with increasing seed MC during threshing in both cultivars.For obtaining high seed quality,indicated by at least 90,85,and 80% of SG,AAG,and CTG,respectively,JK 968 and DD2 should be threshed at MC lower than 15 and 18%,respectively.Furthermore,the damage mainly occurred in the apical part of seeds,irrespective of the threshing MC in both cultivars.When the embryo was damaged,seedling emergence rates were significantly reduced with great influence on shoot length.Damage to the endosperm resulted in little effect on seedling performance.Flint maize DD2 was more tolerant to mechanical threshing than dent maize JK968.These results provided technical reference for the production and processing of high vigor maize seeds.

Cloning and Characterization of Genes Coding for Fructan Biosynthesis Enzymes (FBEs) in Triticeae Plants

Fructan is not only a carbon source for storage but also plays an important role as anti-stress agents in many plant species. Complex fructans having both β-（2,1）- and β-（2,6）-linked fructosyl units accumulate in Triticeae plants commonly. Three enzymes （sucrose： sucrose 1-fructosyltransferase, 1-SST, EC： 2.4.1.99; sucrose： fructan 6-fructosyltransferase, 6- SFT, EC： 2.4.1.10; and fructan： fructan 1-fructosyltransferase, 1-FFT, EC： 2.4.1.100） were involved in fructan biosynthesis in Triticeae plant species. We successfully isolated these genes from tetraploid wheat （Triticum turgidum, genotype： AABB）, common wheat （Triticum aestivum L., genotype： AABBDD） and three wild relatives of common wheat, Triticum urartu Thum. （the origin of the AA genome）, Aegilops speltoides （Tausch） Gren. （the putative source of the SS genome） and Aegilops tauschii Coss. （the source of the DD genome）. Sequence analysis revealed that all the FBEs （fructan biosynthetic enzymes） had three highly conserved functional motifs except 1-SST （EU981912） from tetraploid wheat species only with conserved DPNG. Low pI （isoelectric point） and potential N-glycosylation sites were predicted, which were crucial for protein compartmentation and post-translational process. Analysis on subcelluar localization signals showed that only 6-SFT had vacuolar-directed signal. Sequences alignment result showed that 1-SST and 1-FFT were more conservative and had closer relationship each other, while 6-SFT was more active during the evolution processing. According to the syntenic relationship between wheat and rice genome, FBEs were predicated to be located on the homeologous group 6 and group 2 chromosomes. Expression profile confirmed that expression of all the three FBEs were drought-stress induced. This study can assist to establish a useful theoretical platform for cold- or drought-tolerant improvement of wheat by modulating FBEs expression.

Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS 1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST （glutathione-S-transferase） in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 （DE3）. IPTG （0.5 mmol L-1） was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32p-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses （e.g., salt-） induced signaling pathway.

Isolation and Functional Analysis of the bZIP Transcription Factor Gene TaABP1 from a Chinese Wheat Landrace

In plants, basic leucine zipper （bZIP） transcription factors play important roles in regulatory processes, including stress response, pathogenic defense and light response as well as organ and tissue differentiation. Chinese wheat landrace Pingyaoxiaobaimai （PYXBM）, an original parent of drought tolerant wheat varieties grown in northern China, is significantly tolerant to abiotic stresses such as drought, cold and nutrient deficiencies. In order to isolate key stress-responsive genes and then improve stress tolerances of conventional varieties, a bZIP transcription factor gene was isolated from a cDNA library of drought-treated PYXBM using the in situ plaque hybridization method, and was designated as Triticum aestivum L. abscisic acid （ABA）-responsive element binding protein 1 （TaABP1）. It encodes 372 amino acids, and contains three conserved domains （C1-C3） in the N terminal and a bZIP domain in the C terminal which is a typical protein structure for the group member of bZIP family. Transcriptional activation analysis showed that TaABP1 activated the expression of downstream reporter genes in yeast without ABA application. TaABP1 protein fused with green fluorescent protein （GFP） demonstrated that the localization of TaABP 1 protein is in the nucleus. Expression pattern assays indicated that TaABP1 was strongly induced by ABA, high salt, low temperature and drought, and its expression was stronger in stems and leaves than in the roots of wheat. Furthermore, overexpression of TaABP1 in tobacco showed significant improvement of drought tolerance. Data suggested that TaABP1 may be a good candidate gene for improving stress tolerance of wheat by genetic transformation and elucidation of the role of this gene will be useful for understanding the mechanism underlying drought tolerance of Chinese wheat landrace PYXBM.

Analysis of Indica-Japonica Differentiation in Rice Parents and Derived Lines Using ILP Markers

Revealing the indica-japonica differentiation in parents of hybridization between indica and japonica rice and their derived lines can provide theoretical and practical bases for the breeding of practical inter-subspecific hybrid rice. Using subspeciesspecific molecular markers ILP （intron length polymorphism） and Cheng＇s index, the indica-japonica differentiation was analyzed with special materials including 18 indica-japonica hybrid parents and 39 derived lines, which accumulated different wide compatibility and restoring genes by convergent cross method in 21 years spanning four breeding phases. The indica-japonica differentiation was detected on all tested loci in 57 materials. Among the 18 parental lines, 4 were japonica type, 5 japonicaclinous type, 8 indicaclinous type and one indica type. The japonica proportion indexes in indica restorer lines Minghui 63 and 9308 were 12.50 and 33.33 %, respectively, while that in japonica restorer line C418 was only 31.25%. Among the 39 derived lines from indica-japonica hybridization, one was japonica type, 11 japonicaclinous type, 20 indicaclinous type and 7 indica type. The japonica proportion index in Minghui 502 was only 10.42%. The results of indica and japonica classification by ILP molecular markers and Cheng＇s index were relatively consistent. The correlation coefficient between the japonica proportion index and morphology index was 0.794＊＊, while that between the indica proportion index and morphology index was -0.7662＊＊. ILP markers could be used to accurately detect the proportion of indica/japonica content in the genome of a rice variety. The results of indica-japonica differentiation analysis could make reasonable explanation for that the hybrids obtained from indica-japonica type restorer lines had obvious heterosis. This conclusion would provide important guidance in efficient use of beneficial genes of inter-subspecific hybrid rice.

Isolation and Expression Analysis of a Novel Abiotic Stress-Induced Gene W89 from Wheat

Water stress and cold stress are important factors restricting plant growth. However, there is little knowledge on the function of stress-responsive genes in plants. Therefore, it is necessary to clone some important genes to study the mechanism of plant adaptation to abiotic stress for improvement of plant resistance. A putative water stress-induced gene, W89, was cloned from the eDNA library of drought-treated wheat seedlings by phage hybridization in situ, and its entire length was obtained using 5＇-rapid amplification of eDNA ends （RACE） and reverse transcription-polymerase chain reaction （RT-PCR）. The full-length eDNA of W89 consists of 2 392 bp and contains a 1 896 bp open reading frame （ORF） encoding a 631 amino acid protein. Southern blot analysis indicated that W89 was a single-copy gene. RT-PCR analysis revealed that the expression of W89 was upregulated by drought, cold, and abscisic acid （ABA）. Amino acid sequence analysis discovered that W89 had a conserved region of DUF248 （pfam03141）, which contained a methyltransferase domain with a sterile alpha motif （SAM）-binding motif. Phylogenetic analysis showed that W89 was 66% identical to Oryza sativa dehydration-responsive protein （BAD67956）. It was supposed that W89 was a novel dehydration-responsive protein encoding gene. On the basis of the functions of methyltransferase and the SAM-binding motif, the SAM-binding motif of W89 was supposed to be connected with other proteins or transcription factors to transduce stress signals and finally regulate the expression of stress-responsive genes on the early stage of drought stress.

Tracking and Monitoring Leaf Development, Coupling Law and Regulation Techniques during Flowering Period of Hybrid Foxtail Millet (<i>Setaria italica</i>(L.) P. Beauv.) Parental Lines

The determining factor of Setaria italica (L.) P. Beauv. is the coupling of its flowering stage and outcrossing rate which leads to low and unstable seed yields in self-pollinated foxtail millet hybrids and thereby limits their large-scale application. In this study, Datong 27, Datong 29 and gu 83 were screened and identified through meticulous observations of their pollination habitats. High exposure rate, degree of exposure and plump of stigma are good factors to accept foreign pollen. Datong 27 and Datong 29 have some additional characteristics, such as long filaments and exposed and full anthers that contain a large amount of pollen. We transformed into a series of stigma-exposed and plump sterile lines that easily accepted exotic pollen. New restorer lines with anthers that were full of powder and exhibited quick recovery, which improved the parental lines’ heterosexual characteristics. By tracking and monitoring the leaf development of the new sterile and restorer lines, a coupling law of leaf development was determined and a series of flowering control measures were formulated. These factors ensured that the parental lines encounter one another during the flowering stage. By utilizing fertilizer and water, the vitality of the female stigma, amount of powder scattered and powder loosening time were prolonged, which increased hybrid seed yields from 1500 to 3000 kg/hm2. These findings were helpful in resolving the technical problems of seed production that restricted the propagation of foxtail millet hybrids and supporting future large-scale applications.

Genetic Diversity and Classification of Chinese Elite Foxtail Millet [Setaria italica (L.) P. Beauv.] Revealed by Acid-PAGE Prolamin

Arid and semi-arid regions of China account for more than half of the country. Because of drought resistance and high nutritive value, elite foxtail millet (Setaria Italica (L.) P. Beauv.) is one of the most important cereal crops in China. Evaluation of germplasm and genetic diversity of foxtail millet is still in its infancy, but prolamin could play an important role as a protein marker. To investigate the genetic diversity and population structure of foxtail millet from different ecological zones of China, 90 accessions of foxtail millet were collected from three major ecological areas: North, Northwest, and Northeast China. The prolamin contents were examined by acid polyacrylamide gel electrophoresis (acid-PAGE). Five to twenty-two prolamin bands appeared in tested varieties, of which were polymorphic, so prolamin patterns of foxtail millet varieties can be used in variety identification and evaluation. Structure analysis identified six groups, which matches their pedigree information but not their geographic origins. This indicated a high degree (87.78%) of consistency with a phylogenetic classification based on SSR. The results showed prolamin banding patterns were an effective method for analyzing foxtail millet genetic variability.

Two soybean homologues of TERMINAL FLOWER 1 control flowering time under long day conditions

Flowering time is a key agronomic trait that directly affect the adaptation and yield of soybean.After whole genome duplications,about 75%of genes being represented by multiple copies in soybean.There are four TERMINAL FLOWER 1(TFL1)genes in soybean,and the TFL1b(Dt1)has been characterized as the determinant of stem growth habit.The function of other TFL1 homologs in soybean is still unclear.Here,we generated knockout mutants by CRISPR/Cas9 genome editing technology and found that the tfl1c/tfl1d double mutants flowered significantly earlier than wild-type plants.We investigated that TFL1c and TFL1d could physically interact with the b ZIP transcription factor FDc1 and bind to the promoter of APETALA1a(AP1a).RNA-seq and q RT-PCR analyses indicated that TFL1c and TFL1d repressed the expressions of the four AP1 homologs and delayed the flowering time in soybean.The two genes play important roles in the regulation of flowering time in soybean and mainly act as the flowering inhibitors under long-day conditions.Our results identify novel components in the flowering-time regulation network of soybean and will be invaluable for molecular breeding of improved soybean yield.

Evaluation of allergenic protein profiles in three Chinese high-oleic acid peanut cultivars using NanoLC-Orbitrap mass spectrometry

High oleic-acid peanuts are known for their pre-longed shelf-life and health benefit due to high content of oleic fatty acid.However,the allergenicity and allergenic protein profiles in Chinese high-oleic peanuts have yet to be studied.For this purpose,an Orbitrap Fusion mass spectrometry(MS)-based method that is feasible for identification of putative allergenic protein as well as semi-quantitation of five major allergen protein in three different Chinese high-oleic peanut cultivars(JH 13,JH 16 and JH 18)have been reported.Results show that three Chinese high-oleic acid peanut cultivars selected all contained highly allergenic proteins Ara h1,Ara h 2,Ara h 3 and Ara h 6.The allergenic protein profiles of Chinese high-oleic acid peanut cultivars were very similar to that of conventional peanuts,but the allergenic protein subunits varied greatly among higholeic peanuts.Additionally,a comprehensive peptide-filtering pipeline had been developed for identification of potential peptide markers in peanut allergen proteins.Through the peptide-filtering pipeline,three novel peptide markers,IVQIEAKPNTLVLPK,SSNPDIYNPQAGSLR and AQSENYEYLAFK surrogate to Ara h 1,Ara h 3 with high abundance,good MS response and highly reliability were identified,which can be used as candidate peptide markers for the detection of peanut allergens in different food matrices.

Primary metabolite contents are correlated with seed protein and oil traits in near-isogenic lines of soybean

Soybean(Glycine max[L.]Merr.)is an important source of human dietary protein and vegetable oil.A strong negative correlation between protein and oil contents has hindered efforts to improve soybean seed quality.The metabolic and genetic bases of soybean seed composition remain elusive.We evaluated metabolic diversity in a soybean near-isogenic line(NIL)population derived from parents(JD12 and CMSD)with contrasting seed oil contents.Using GC-TOF/MS,we compared seed primary metabolites of high protein/low oil lines,low protein/high oil lines,and their parents.Principal-components analysis showed that metabolic profiles of all progeny lines could be discriminated based on protein and oil contents.Univariate analysis revealed wide variation and transgressive segregation of metabolites in the population.Twenty-eight annotated metabolites,in particular free asparagine,free 3-cyanoalanine,and L-malic acid,were correlated with seed protein content or seed oil content or seed protein and oil content.These results shed light on the metabolic and genetic basis of soybean seed composition.

Molecular Characterization of a Triticum durum-Haynaldia villosa Amphiploid and Its Derivatives for Resistance to Gaeumannomyces graminis var. tritici

Take-all is a serious disease found in wheat across the world. Haynaldia villosa is considered to be resistant to take-all at a high level. TH3 was an amphiploid （2n =42, AABBVV） between Triticum durum and Haynaldia viUosa with significant resistance to take-all fungus isolated from China. In greenhouse experiment, the derivatives of the hybrid between wheat and TH3 showed better resistance to take-all than that of the wheat control. One of the derivatives named HW918-5 was selected for further analysis. Cytological and genomic in situ hybridization （GISH） analysis indicated that a monotelosome originated from H. villosa existed in the genome of the offspring of the line HW918-5. The monotelosome with promising resistant gene for take-all was located on the 3V chromosome of H. villosa in the further PCR-based molecular analysis.

Evaluation and Identification of Resistance to Diseases and Pests for Rice Varieties in Hainan

[Objectives]This study was conducted to select multi-resistant rice varieties in Hainan.[Methods]Seven new rice germplasms were identified by naturally induced disease nursery,field spraying and artificial inoculation.[Results]The restorer line Haihui 1558 was resistant to the dominant races ZB and ZC of rice blast and Tryporyza incertulas,and moderately resistant to pathotype IV of bacterial blight.The restorer line Haihui 227 was resistant to pathotype IV of bacterial blight and T.incertulas.Red rice Hainonghong 1 was resistant to pathotype IV of bacterial blight,and moderately resistant to the dominant races ZB and ZC of rice blast.Black glutinous rice Haifengheinuo 2 was moderately resistant to pathotypes IV of bacterial blight and T.incertulas.[Conclusions]The study provides reference for utilization of rice breeding on resistance to diseases and pests.

Breeding of Rice Restorer Line Haihui 818 of Multi-resistant Gene Pyramiding

Haihui 818 is a new restorer line by cross breeding with Hua 23 S and Huahui 8131.Good plant type,high tillering ability,fine grain quality,strong restoring ability and high yield were shown in hybrid production.After artificial inoculation and field stress and molecular market-assisted selection,the results showed that rice-blast resistance genes Pi1,BB resistance genes Xa7,and brown planthopper resistance genes Bph3,Bph14 and Bph15 were pyramided together.Among its hybrids,Bo II You 818(Bo II A/Haihui 818)was approved for commercial production by Hainan Provincial Crop Variety Approval Committee in 2015.

Sequence Mutation of Exon 7 of Badh2 Gene and Aroma Identification of Fragrant Rice Varieties

In this study,we performed amplificaion and sequence analysis of exon7 of gene Badh2 of 12 fragrant rice materials,and identified the aroma of fragrant rice materials by the method of seed chewing and KOH soaking,so as to analyze the sequence mutation of exon 7 in the Badh2 gene of rice material and its corresponding relation with the flavor character.The results showed that an 8 bp deletion(aaaa--t---ggc)and a mutation of SNP(g→t)in exon 7 of Badh2 gene were found in 10 materials,including Xiangnuo,Lvjinxiang,Meixiangzhan 2,Huaxiang,Yuexiangxuan 1,Hongyuxiang,Meixiangxuan 1,Baxiangxuan 1,Taixiangxuan 1,Taixiangxuan 2.This mutation was consistent with the mutation of EU155083 sequence in GenBank and was reported for the first time in Chinese rice materials.In these 10 fragrant rice materials with mutation,Huaxiang and Meixiangxuan 1 were identified as the heterozygote genotype,and Hongyuxiang was identified as non-fragrant rice,so the sequence mutation in exon 7 of Badh2 gene in fragrant rice materials did not correspond to aroma traits one by one;and 7 materials were identified as fragrant rice,and the brown rice of Meixiangzhan 2 and Yuexiangyuan 1 had sweet taste.The results could provide a reference for the research on the genetic mechanism of rice aroma character and the promotion of fragrant rice varieties.

Fine mapping of a major QTL qHYF_B06 for peanut yield

High yield is a major objective for peanut(Arachis hypogaea L.) breeding worldwide. However, fewer yield-related quantitative trait loci(QTL) have been reported in peanut than in other staple food crops such as rice(Oryza sativa), wheat(Triticum aestivum), and maize(Zea mays). This study aimed to identify stable major-effect QTL associated with pod yield per plant, hundred-pod weight for double-seeded pods,hundred-seed weight, shelling percentage, and pod number per plant, allowing us to predict candidate genes by means of transcriptome and genome sequencing. To this end, we used a population of recombinant inbred lines comprising 192 F9:11families derived from a JH6 × KX01-6 cross to construct a highresolution genetic map(1705.7 c M) consisting of 2273 polymorphic SNPs, with 0.75 c M(on average)between adjacent SNPs. We identified two high-confidence, yield-related QTL, qHYF_A08 and qHYF_B06, explaining 5.78%–31.40% of phenotypic variation and with LOD values of 5.10–24.48, in six environments. qHYF_A08 mainly explained the variation in shelling percentage, whereas qHYF_B06explained variation in hundred-pod weight and hundred-seed weight and accounted for 8.77%–31.40%of the variation in effective pod number per plant, pod number per plant, and shelling percentage. We narrowed down qHYF_B06 to an 890-kb interval using an advanced mapping population.Transcriptome and genome analyses revealed that only Arahy.129FS0 and Arahy.3R9A5K in the candidate mapping interval were differentially expressed between JH6 and KX01-6, with substantial structural variations in their promoter and coding regions. Genotypes of 208 peanut accessions determined using a diagnostic CAPS marker suggested that the two haplotypes of Arahy.3R9A5K were highly associated with hundred-seed weight and hundred-pod weight;this diagnostic CAPs marker could therefore be useful for selecting high-yielding lines during peanut breeding. Overall, our results provide valuable information for cloning alleles with favorable effects on peanut yield.

Transcriptome Analysis of oserf922 Mutants Reveals New Insights into Rice Blast Resistance

Rice blast disease,caused by fungal Magnaporthe oryzae,severely threatens food security.The susceptibility(S) genes have emerged to support pathogenesis,and disruption of S gene usually confers increased resistance to multiple pathogen isolates.Rice ethylene response factor gene Os ERF922 is a potential S gene in blast disease.However,how Os ERF922 negatively regulates resistance against M.oryzae is still unknown.

Establishment of a Highly Efficient Regeneration System for the Mature Embryo Culture of Wheat

Establishment of a highly efficient regeneration system for the mature embryo of wheat will provide a convenient tool for wheat tissue culture and transformation, thereby facilitating the transformation of foreign genes into wheat. By using the mature embryos derived from 20 different wheat lines including Shi 4185, Yumai 66, Lunxuan 987, CB037, Yangmai 6, Xinchun 9, Bobwhite, Han 6172, Zheng 9023, Jimai 20, Ningchun 4, and Jing 411, the effects of some factors including inoculation methods, initiating culture media, organic additives, antioxidants, and auxins on the regeneration from the explants were evaluated. The results indicated that the scraping embryo culture was better than the whole embryo culture, the Aa medium was better than the SD2 medium and dicamba was better than 2,4-D in increasing the regeneration frequency. An Adi medium was established in this study by adding silver nitrate, cysteine, ascorbic acid, dicamba, glutamine into the Aa medium at the concentration of 4,40, 100, 2, and 5 mg L^-1, respectively. By using the Adi medium and the scraping technique, the regeneration frequencies of the mature embryos of CB037, Lunxuan 987, Hart 6172, Yangmai 6, Bobwhite, Zheng 9023, Shi 4 185, and Jimai 20 became 85.6, 60,1, 46.0, 42.1,42.0, 34.0, 33.0, and 32.0%, respectively, which were about 5-8 times higher than that obtained from the conventional culture mediums and techniques. This novel regeneration system could be helpful in wheat transformation.

Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

HrcQ is necessary for Xanthomonas oryzae pv. oryzae HR-induction in non-host tobacco and pathogenicity in host rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae(Xoo), is one of the most destructive diseases of rice(Oryza sativa L.) worldwide. The type III secretion system(T3SS) of Xoo, encoded by the hrp(hypersensitive response and pathogenicity) genes, plays critical roles in conferring pathogenicity in host rice and triggering a hypersensitive response(HR) in non-host plants. To investigate the major genes conferring the pathogenicity and avirulence of Xoo, we previously constructed a random Tn5-insertion mutant library of Xoo strain PXO99A. We report here the isolation and characterization of a Tn5-insertion mutant PXM69. Tn5-insertion mutants were screened on indica rice JG30, which is highly susceptible to PXO99A, by leaf-cutting inoculation.Four mutants with reduced virulence were obtained after two rounds of screening. Among them, the mutant PXM69 had completely lost virulence to the rice host and ability to elicit HR in non-host tobacco. Southern blotting analysis showed a single copy of a Tn5-insertion in the genome of PXM69. PCR walking and sequencing analysis revealed that the Tn5 transposon was inserted at nucleotide position 70,192–70,201 in the genome of PXO99A, disrupting the type III hrc(hrp-conserved) gene hrcQ, the first gene in the D operon of the hrp cluster in Xoo. To confirm the relationship between the Tn5-insertion and the avirulence phenotype of PXM69, we used the marker exchange mutagenesis to create a PXO99Amutant, ΔhrcQ::KAN, in which the hrcQ was disrupted by a kanamycin-encoding gene cassette at the same site as that of the Tn5-insertion. ΔhrcQ::KAN showed the same phenotype as mutant PXM69. Reintroduction of the wild-type hrcQ gene partially complemented the pathogenic function of PXM69. RT-PCR and cellulase secretion assays showed that the Tn5-disruption of hrcQ did not affect transcription of downstream genes in the D operon and function of the type II secretion system. Our results provide new insights into the pathogenic functions of clustered hrp genes in Xoo.