A novel simple extracellular leucine-rich repeat (eLRR) domain protein from rice (OsLRR1) enters the endosomal pathway and interacts with the hypersensitive-induced reaction protein 1 (OsHIR1)

Receptor-like protein kinases (RLKs) containing an extracellular leucine-rich repeat (eLRR) domain, a transmembrane domain and a cytoplasmic kinase domain play important roles in plant disease resistance. Simple eLRR domain proteins structurally resembling the extracellular portion of the RLKs may also participate in signalling transduction and plant defence response. Yet the molecular mechanisms and subcellular localization in regulating plant disease resistance of these simple eLRR domain proteins are still largely unclear. We provided the first experimental evidence to demonstrate the subcellular localization and trafficking of a novel simple eLRR domain protein (OsLRR1) in the endosomal pathway, using both confocal and electron microscopy. Yeast two-hybrid and in vitro pull-down assays show that OsLRR1 interacts with the rice hypersensitive-induced response protein 1 (OsHIR1) which is localized on plasma membrane. The interaction between LRR1 and HIR1 homologs was shown to be highly conserved among different plant species, suggesting a close functional relationship between the two proteins. The function of OsLRR1 in plant defence response was examined by gain-of-function tests using transgenic Arabidopsis thaliana . The protective effects of OsLRR1 against bacterial pathogen infection were shown by the alleviating of disease symptoms, lowering of pathogen titres and higher expression of defence marker genes.     

Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>) inbred line,Kenshin

We developed a transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin, with high tolerance to soft rot disease. Tolerance was conferred by expression of N-acyl-homoserine lactonase (AHL-lactonase) in Chinese cabbage through an efficient Agrobacterium-mediated transformation method. To synthesize and express the AHL-lactonase in Chinese cabbage, the plant was transformed with the aii gene (AHL-lactonase gene from Bacillus sp. GH02) fused to the PinII signal peptide (protease inhibitor II from potato). Five transgenic lines were selected by growth on hygromycin-containing medium (3.7% transformation efficiency). Southern blot analysis showed that the transgene was stably integrated into the genome. Among these five transgenic lines, single copy number integrations were observed in four lines and a double copy number integration was observed in one transgenic line. Northern blot analysis confirmed that pinIISP-aii fusion gene was expressed in all the transgenic lines. Soft rot disease tolerance was evaluated at tissue and seedling stage. Transgenic plants showed a significantly enhanced tolerance (2–3-fold) to soft rot disease compared to wild-type plants. Thus, expression of the fusion gene pinIISP-aii reduces susceptibility to soft rot disease in Chinese cabbage. We conclude that the recombinant AHL-lactonase, encoded by aii, can effectively quench bacterial quorum-sensing and prevent bacterial population density-dependent infections. To the best of our knowledge, the present study is the first to demonstrate the transformation of Chinese cabbage inbred line Kenshin, and the first to describe the effect of the fusion gene pinIISP-aii on enhancement of soft rot disease tolerance.     

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5 r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 9 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in C 90%reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.     

Overexpression of wheat lipid transfer protein gene TaLTP5 increases resistances to Cochliobolus sativus and Fusarium graminearum in transgenic wheat

The fungus Cochliobolus sativus is the main pathogen of common root rot, a serious soil-borne disease of wheat (Triticum aestivum L.). The fungus Fusarium graminearum is the primary pathogen of Fusarium head blight, a devastating disease of wheat worldwide. In this study, the wheat lipid transfer protein gene, TaLTP5, was cloned and evaluated for its ability to suppress disease development in transgenic wheat. TaLTP5 expression was induced after C. sativus infection. The TaLTP5 expression vector, pA25-TaLTP5, was constructed and bombarded into Chinese wheat variety Yangmai 18. Six TaLTP5 transgenic wheat lines were established and characterized. PCR and Southern blot analyses indicated that the introduced TaLTP5 gene was integrated into the genomes of six transgenic wheat lines by distinct patterns, and heritable. RT-PCR and real-time quantitative RT-PCR revealed that the TaLTP5 gene was over-expressed in the transgenic wheat lines compared to segregants lacking the transgene and wild-type wheat plants. Following challenge with C. sativus or F. graminearum, all six transgenic lines overexpressing TaLTP5 exhibited significantly enhanced resistance to both common root rot and Fusarium head blight compared to the untransformed wheat Yangmai 18.     

Activation of defense responses in Chinese cabbage by a nonhost pathogen, Pseudomonas syringae pv. tomato

Pseudomonas syringae pv. tomato (Pst) causes a bacterial speck disease in tomato and Arabidopsis. In Chinese cabbage, in which host-pathogen interactions are not well understood, Pst does not cause disease but rather elicits a hypersensitive response. Pst induces localized cell death and H2O2 accumulation, a typical hypersensitive response, in infiltrated cabbage leaves. Pre-inoculation with Pst was found to induce resistance to Erwinia carotovora subsp. carotovora, a pathogen that causes soft rot disease in Chinese cabbage. An examination of the expression profiles of 12 previously identified Pst-inducible genes revealed that the majority of these genes were activated by salicylic acid or BTH; however, expressions of the genes encoding PR4 and a class IV chitinase were induced by ethephon, an ethylene-releasing compound, but not by salicylic acid, BTH, or methyl jasmonate. This implies that Pst activates both salicylate-dependent and salicylate-independent defense responses in Chinese cabbage.     

软腐白菜细菌群落结构多样性与生长环境的相关性

[目的]通过对比分析不同生境白菜软腐病变组织与根系土壤相关细菌的群落结构,探讨白菜软腐细菌种群的多样性,以及与生境土壤细菌种群的相关性.[方法]样品采自河南省2个不同生态型白菜田,以成熟白菜软腐组织及病株根系土壤为目标,利用模拟原环境的培养基成分和条件,对样品中的细菌进行高通量分离培养和细胞16S rRNA基因序列比对分析,获得各样品细菌种群结构及其丰度,进而对各样品的优势菌群进行对比分析.[结果]两种不同生境白菜软腐组织细菌总量M05T为4.0×l0s cell/g、Q2T为1.2×1011 cell/g,分别获得纯菌56株和85株.M05T优势菌为萎蔫短小杆菌萎蔫亚种(Curtobacterium flaccunfaciens pv.Flaccumfaciens);Q2T优势菌为假单胞菌(Pseudomonas spp.)(柄木槿假单胞菌(P.hibiscicola)、台湾假单胞菌(P.taiwanensis)、托木尔假单胞菌(P.tuomuerensis)、莫塞尔假单胞菌(P.mosselii)).根系土壤细菌总量M05S为2.7 × 105 cell/g、Q2S为6.2 × 107 cell/g,分别获得纯菌36株和70株.M05S优势菌为巨大芽胞杆菌(Bacillus megatherium);Q2S优势菌为假单胞菌(Pseudomonas spp.)(香鱼假单胞菌(P.plecoglossicida)、栖木槿假单胞菌(P.hibiscicola)、类黄色假单胞菌(P.parafulva)、蒙氏假单胞菌(P.monteilii)、膝形假单胞菌(P.geniculata)).[结论]依据不同生境的白菜软腐组织和根系土壤细菌群落结构对比分析,认为白菜软腐菌可能具有多样性和多种致病来源,本研究为软腐病多种防治措施的制定提供基础研究和菌种资源.     

Generation of Chinese cabbage resistant to bacterial soft rot by heterologous expression of <Emphasis Type="Italic">Arabidopsis WRKY75</Emphasis>

Soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is a serious disease in Chinese cabbage (Brassica rapa L. subsp. pekinensis). To reduce the severity of soft rot symptoms in Chinese cabbage, Arabidopsis AtWRKY75 was introduced into Chinese cabbage by Agrobacterium-mediated transformation, which was previously reported to reduce susceptibility to Pcc infection in Arabidopsis. Three independent Chinese cabbage transgenic lines carrying AtWRKY75 were obtained. The growth phenotypes of AtWRKY75 overexpression (OE) lines were normal. Bacterial soft rot symptoms and Pcc growth were reduced in AtWRKY75-OE Chinese cabbage lines compared with WT plants. In contrast, overexpression of AtWRKY75 had no effect on infection with a hemibiotrophic pathogen, Xanthomonas campestris pv. campestris (Xcc) causing black rot disease. These results are consistent with those observed in the transgenic Arabidopsis. We found that AtWRKY75 activated a subset of Chinese cabbage genes related to defense against Pcc infection, such as Meri15B, BrPR4, and BrPDF1.2 (but not BrPGIP2). Moreover, overexpression of AtWRKY75 caused H₂O₂ production and activation of H₂O₂ scavenge enzyme genes, suggesting that H₂O₂ played a role in AtWRKY75-mediated resistance to Pcc. Together, these results demonstrated that AtWRKY75 decreased the severity of Pcc-caused bacterial soft rot and activated a subset of Pcc infection defense-related genes in Chinese cabbage similar to in Arabidopsis. It is suggested that AtWRKY75 is a candidate gene for use in crop improvement, because it results in reduced severity of disease symptoms without concurrent growth abnormalities.     

The Sweet Pepper Ferredoxin-Like Protein (pflp) Conferred Resistance Against Soft Rot Disease in Oncidium Orchid

Genetic engineering to date has not been used to introduce disease resistance genes into the orchid gene pool. The ferredoxin-like protein gene originally isolated from sweet pepper is thought to function as a natural defense against infection due to its antimicrobial properties. Hence it was reasoned that introduction of this gene might produce Oncidium plants resistant to Erwinia carotovora, the causal agent for the soft rot disease. An expression vector containing sweet pepper ferredoxin-like protein (pflp) cDNA, hph and gusA coding sequence was successfully transformed into protocorm-like bodies (PLBs) of Oncidium orchid, using Agrobacterium tumefaciens strain EHA105. A total of 17 independent transgenic orchid lines was obtained, out of which six transgenic lines (-glucuronidase (GUS) positive) were randomly selected and confirmed by Southern, northern and western blot analyses. A bioassay was conducted on the transgenic lines. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest that pflp may be an extremely useful gene for genetic engineering strategies in orchids to confer resistance against soft rot disease.     

CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum

Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum.     

Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.     

大白菜骨干自交系的苗期抗病性评价

为明确大白菜骨干自交系的抗病性,本研究于2012-2014年,对课题组保存和创制的203个大白菜自交系进行了霜霉病、病毒病、黑腐病、黄萎病和根肿病的苗期抗性评价。结果显示,高抗上述病害的自交系分别有7、9、0、31和12个;只抗其中一种病害的自交系82个;兼抗两种病害的有61个,兼抗三种病害的自交系有28个,兼抗四种病害的自交系有4个。自交系11-234、04-622、12-85、13-108和09-894综合抗病性最优。此外,春大白菜、夏大白菜和秋大白菜三种生态类群间,以及四种叶球抱合类群间的抗病性表现出明显差异。     

The products of the broken Tm-2 and the durable Tm-2(2) resistance genes from tomato differ in four amino acids

To gain an insight into the processes underlying disease resistance and its durability, the durable Tm-2(2) resistance gene was compared with the broken Tm-2 resistance gene. The Tm-2 gene of tomato could be isolated via PCR with primers based on the Tm-2(2) sequence. The Tm-2 gene, like the Tm-2(2) gene, encodes an 861 amino acid polypeptide, which belongs to the coiled coil/nucleotide binding site/leucine-rich repeat class of resistance proteins. The functionality and the nature of the isolated Tm-2 gene were confirmed by introducing the gene under the control of the 35S promoter into tomato mosaic virus-susceptible tobacco. This transgenic tobacco was crossed with transgenic tobacco plants producing the movement protein (MP)-authenticated MP as the Avr protein of the Tm-2 resistance. The Tm-2(2) and Tm-2 open reading frames only differ in seven nucleotides, which on a protein level results in four amino acid differences, of which two are located in the nucleotide binding site and two are located in the leucine-rich repeat domain. The small difference between the two proteins suggests a highly similar interaction of these proteins with the MP, which has major implications for the concept of durability. Comparison of the two resistance-conferring alleles (Tm-2 and Tm-2(2)) with two susceptible alleles (tm-2 and lptm-2) allowed discussion of the structure-function relationship in the Tm-2 proteins. It is proposed that the Tm-2 proteins display a partitioning of the leucine-rich repeat domain, in which the N-terminal and C-terminal parts function in signal transduction and MP recognition, respectively.     

Xa26, a gene conferring resistance to Xanthomonas oryzae pv. oryzae in rice, encodes an LRR receptor kinase-like protein

Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious rice diseases worldwide. A rice gene, Xa26, conferring resistance against Xoo at both seedling and adult stages was isolated by map-based cloning strategies from the rice cultivar Minghui 63. Xa26 belongs to a multigene family consisting of four members. It encodes a leucine-rich repeat (LRR) receptor kinase-like protein and is constitutively expressed. Sequence analysis revealed that IRBB3 and Zhachanglong lines that are resistant to a broad range of Xoo strains, also carry Xa26. However, significant difference in lesion length was observed among these lines after inoculation with a set of Xoo strains. Moreover, transgenic plants carrying Xa26 showed enhanced resistance compared with the donor line of the gene in both seedling and adult stages. These results suggest that the resistance conferred by Xa26 is influenced by the genetic background.     

Induced suppression of soft rot disease in tobacco by combined application of Bacillus subtilis strain B4 and chemical elicitor BTH

In this study, the utility of the combined application of Benzo-(1,2,3)-thidiazole-7-carbothioic acid S-methyl ester (BTH), a systemic acquired resistance (SAR) inducer, and the plant growth-promoting rhizobacteria (PGPR), Bacillus subtilis strain B4 (B4), was investigated for the suppression of soft rot disease caused by Pectobacterium carotovorum SCC1 (SCC1) in tobacco seedlings. Soft rot disease was completely suppressed in tobacco with combined application of B4 strain and 0.1 mM BTH when compared to individual application upon pathogen challenge. In addition, the population of bacterial cells of B4 strain was found to be greater when cultured in growth media in the presence of BTH, compared to the growth of B4 strain in the absence of BTH. Spectrophotometer analysis revealed that there was an increased broad range of compounds in the culture filtrate of B4 strain when grown with BTH, compared to the culture filtrate of B4 strain alone. The combined application of B4 strain and 0.1 mM BTH induced the increased expression of PR1a::GUS on tobacco and elicited systemic resistance against SCC1 when compared to individual application. However, there was low expression of PR1a::GUS in the water-treated control. Hence, the integrated use of BTH and B4 strain might be one of the strategies for biological control of soft rot disease through induced systemic resistance (ISR) in tobacco plants.     

Antisense Expression of a NBS-LRR Sequence in Sunflower (Helianthus annuus L.) and Tobacco (Nicotiana tabacum L.): Evidence for a Dual Role in Plant Development and Fungal Resistance

A partial sunflower cDNA clone, PLFOR48, segregating with a resistance marker to Plasmopara halstedii, the causal agent of downy mildew, has been cloned from the mildew resistant sunflower line, RHA 266. PLFOR48 encodes a putative protein with a nucleotide-binding site and a leucine-rich repeat domain, showing significant homology with previously cloned resistance genes belonging to the TIR-NBS-LRR family. Southern blot analysis of non-transgenic sunflower suggests that PLFOR48 is part of a multigenic family. The potential role of PLFOR48 sequence in sunflower resistance to mildew was studied, by assessing loss of function, using expression of the antisense cDNA in RHA 266 sunflower line. Quite unexpectedly, transgenic sunflower lines displayed severe developmental abnormalities, and in particular, on the main meristems of homozygote T2 progeny, thus hampering any further challenge inoculation with Plasmopara halstedii. The presence of homologous sequences to PLFOR48 in Nicotiana tabacum var Samsun NN, as demonstrated by Southern blotting, drove us to consider tobacco as an additional model to investigate the potential role of this sequence in fungal resistance. Expression of the same antisense cDNA in transgenic tobacco lines gave rise to higher degree of susceptibility to Phytophthora parasitica, as well as to severe alterations in seed development. These results suggest that PLFOR48 and homologous sequences could be involved in both regulating developmental pathways and controlling resistance to fungal pathogens.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">OsBISERK1</Emphasis>, a gene encoding a leucine-rich repeat receptor-like kinase,during disease resistance responses in rice

A rice gene, OsBISERK1, encoding a protein belonging to SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) type of leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. The OsBISERK1 encodes a 624 aa protein with high level of identity to known plant SERKs. OsBISERK1 contains a hydrophobic signal peptide, a leucine zipper, and five leucine-rich repeat motifs in the extracellular domain; the cytoplasmic region carries a proline-rich region and a single transmembrane domain, as well as a conserved intracellular serine/threonine protein kinase domain. OsBISERK1 has a low level of basal expression in leaf tissue. However, expression of OsBISERK1 was induced by treatment with benzothiadiazole (BTH), which is capable of inducing disease resistance in rice, and also up-regulated after inoculation with Magnaporthe grisea in BTH-treated rice seedlings and during incompatible interaction between a blast-resistant rice genotype and M. grisea. The results suggest that OsBISERK1 may be involved in disease resistance responses in rice.     

Host-induced gene silencing of Pectobacterium carotovorum quorum sensing gene enhances soft rot disease resistance in potato plants

Quorum sensing is a regulatory mechanism of gene expression in bacteria which regulated many physiological processes such as production of virulence factors in Pectobacterium carotovorum. In this research, the plant expression vector, pBI121, harbouring the anti-quorum sensing gene, aiiA, was transformed into Potato (Solanum tuberosum cv. Agria) explants by Agrobacterium mediated transformation procedure and putative transformants containing pBI121/aiiA were obtained. The results of bioassay test revealed that the expression of aiiA gene in potato plant induces the resistance to the early stage of bacterium pathogenesis. The soft rot symptoms such as wilting, yellowing and tissue maceration were not observed until 48?hr after inoculation of transgenic potatoes with P. carotovorum. Although in non-transgenic plants, the disease symptoms were appeared after 24?hr of plants infection. Hence, this study proves that the heterologous expression of aiiA gene enhances the resistance against potato soft rot disease.     

Pectobacterium carotovorum elicits plant cell death with DspE/F but the P. carotovorum DspE does not suppress callose or induce expression of plant genes early in plant-microbe interactions

The broad-host-range bacterial soft rot pathogen Pectobacterium carotovorum causes a DspE/F-dependent plant cell death on Nicotiana benthamiana within 24 h postinoculation (hpi) followed by leaf maceration within 48 hpi. P. carotovorum strains with mutations in type III secretion system (T3SS) regulatory and structural genes, including the dspE/F operon, did not cause hypersensitive response (HR)-like cell death and or leaf maceration. A strain with a mutation in the type II secretion system caused HR-like plant cell death but no maceration. P. carotovorum was unable to impede callose deposition in N. benthamiana leaves, suggesting that P. carotovorum does not suppress this basal immunity function. Within 24 hpi, there was callose deposition along leaf veins and examination showed that the pathogen cells were localized along the veins. To further examine HR-like plant cell death induced by P. carotovorum, gene expression profiles in N. benthamiana leaves inoculated with wild-type and mutant P. carotovorum and Pseudomonas syringae strains were compared. The N. benthamiana gene expression profile of leaves infiltrated with Pectobacterium carotovorum was similar to leaves infiltrated with a Pseudomonas syringae T3SS mutant. These data support a model where Pectobacterium carotovorum uses the T3SS to induce plant cell death in order to promote leaf maceration rather than to suppress plant immunity.