Peroxidase isoenzymes in the defense response of Capsicum annuum to Phytophthora capsici

Quantitative and qualitative changes in isoperoxidase patterns from stems of three cultivars of pepper ( Capsicum annuum L.). one susceptible, one intermediate and one resistant, were found upon inoculation with Phytophthora capsici using a decapitation method. The peroxidase activity was determined in the intercellular fluid as well as in the cytosolic fraction of the necrotic, healthy and intermediate zones of stems of the three cultivars, 6 days after inoculation. In the intercellular fluid, peroxidase activity of the susceptible cv. Yolo Wonder increased somewhat from 4.7 (healthy zone) to 12.9 (intermediate zone) μmol mg⁻¹ protein min⁻¹, whereas in the intermediate cv. Americano, the peroxidase activity decreased from 123 (healthy zone) to 78 (intermediate zone) μmol mg⁻¹ protein min⁻¹. The most dramatic increase (5.7 to 662 μmol mg⁻¹ protein min⁻¹) in intercellular peroxidase activity was found in the resistant cv. Smith-5. This, in conjunction with the appearance of an additional acidic isoperoxidase (pI 4.4) specific for the cv. Smith-5, could be the reason for the resistance of this cultivar against the fungus attack. The release of peroxidase into the intercellular space as a defense reaction was confirmed by histochemical analysis, showing that peroxidase activity occurred in the intercellular spaces of those stems of the resistant cultivar that had not yet been invaded by the fungus, but was detected neither in the other cultivars nor in the intercellular spaces of such stems of the intermediate and susceptible cultivars that contained growing mycelium of P. capsici. The lack of staining in the intercellular spaces of the susceptible cultivars could be attributed to their low content in peroxidase.     

漾濞大泡核桃WRKY转录因子基因JsWRKY1的克隆及表达特性分析

WRKY转录因子普遍存在于植物体内,在植物的抗病防御反应中起重要作用。本实验基于漾濞大泡核桃（Juglans sigillata）中编码WRKY转录因子的EST序列设计引物,采用快速扩增cDNA末端技术,克隆得到一个新的脓Ky基因的全长cDNA序列,命名为JsWRKY1（KJ170895）。JsWRKY1的cDNA全长为1012bp,含有564bp的开放阅读框,154bp 5’-非翻译区以及294bp的3＇-非翻译区,编码具有187个氨基酸的蛋白质。JsWRKY1编码的氨基酸序列与已知植物WRKY家族成员间的同源性和聚类分析表明JsWRKY1与来源于可可树（Theobroma cacao）和大豆（Glycinemax）中的wRKY相似性较高,属于IIc类wRKY。qRT-PCR分析结果显示,信号分子水杨酸、茉莉酸、H2O2和乙烯处理可以不同程度地诱导漾濞大泡核桃叶片中JsWRKY1的表达。此外,接种胶孢炭疽菌后JsWRKY1的表达量迅速上升,在接种后4h时达到最高水平,之后表达量逐渐下降,暗示JsWRKY1参与漾濞大泡核桃抗胶孢炭疽菌的防卫反应。     

大麦黄花叶病相关基因WRKY55的克隆及功能研究

大麦黄花叶病是我国长江中下游地区大麦种植区域的主要病毒病害,由大麦黄花叶病毒(BaYMV,Barley yellow mosaic virus)及大麦和性花叶病毒(BaMMV,Barley mild mosaic virus)引起,自然条件下病毒寄生于禾谷多黏菌中,通过感染大麦根部导致病害发生。本研究通过分析RNA-seq数据,获得感染BaMMV后上调表达的基因HORVU1Hr1G069640(WRKY55)。荧光定量PCR分析发现WRKY55在感病品种中的表达水平极显著高于抗病材料,说明WRKY55参与到大麦感染黄花叶病的过程。WRKY55全长870 bp,在415～594位核苷酸之间含有WRKY保守结构域,系统进化分析显示该基因位于独立的进化分枝。组织表达分析发现该基因主要在根部和幼嫩组织中表达,而其他成熟部位表达较少甚至没有。农杆菌介导的烟草亚细胞定位实验发现WRKY55位于整个细胞。酵母转录因子活性分析实验未检测到转录激活活性,酵母双杂交实验未检测到WRKY55与感病基因编码蛋白eIF4E和PDIL5-1存在物理相互作用。这些结果显示WRKY55参与到大麦黄花叶病感染。     

Suppression of CaCYP1, a novel cytochrome P450 gene, compromises the basal pathogen defense response of pepper plants

A putative cytochrome P450 gene from chili pepper, Capsicum annuum L. Bukang cytochrome P450 (CaCYP1), was identified using cDNA microarray analysis of gene expression following induction of the leaf hypersensitive response by inoculation of pepper plants with the non-host pathogen Xanthomonas axonopodis pv. glycines 8ra. The full-length cDNA of CaCYP1 encoded a protein of 514 amino acid residues, which contained a putative hydrophobic membrane anchoring domain in the N-terminal region, and a heme-binding motif in the C-terminal region. Analysis of the deduced amino acid sequence of CaCYP1 revealed that it has high homology to Arabidopsis CYP89A5, the function of which is unknown. Expression of CaCYP1 was preferentially increased in pepper plants in response to non-host pathogen inoculation and also during the host resistance response. CaCYP1 expression also increased following treatment with salicylic acid and abscisic acid, while treatment with ethylene had a mild effect. Using a virus-induced gene silencing-based reverse genetics approach, we demonstrated that suppression of CaCYP1 results in enhanced susceptibility to bacterial pathogens. Interestingly, gene silencing of CaCYP1 in pepper plants resulted in the reduced expression of the defense-related genes CaLTP1, CaSIG4, and Cadhn. Our results indicated that CaCYP1, a novel cytochrome P450 in pepper plants, may play a role in plant defense response pathways that involve salicylic acid and abscisic acid signaling pathways.     

Infection of Arabidopsis with a necrotrophic pathogen,Botrytis cinerea,elicits various defense responses but does not induce systemic acquired resistance (SAR)

Botrytis cinerea is a non-specific necrotrophic pathogen that attacks more than 200 plant species. In contrast to biotrophs, the necrotrophs obtain their nutrients by first killing the host cells. Many studies have shown that infection of plants by necrosis-causing pathogens induces a systemic acquired resistance (SAR), which provides protection against successive infections by a range of pathogenic organisms. We analyzed the role of SAR in B. cinerea infection of Arabidopsis. We show that although B. cinerea induced necrotic lesions and camalexin biosynthesis, it did not induce SAR-mediated protection against virulent strains of Pseudomonas syringae, or against subsequent B. cinerea infections. Induction of SAR with avirulent P. syringae or by chemical treatment with salicylic acid (SA) or benzothiadiazole also failed to inhibit B. cinerea growth, although removal of basal SA accumulation by expression of a bacterial salicylate hydroxylase (NahG) gene or by infiltration of 2-aminoindan-2-phosphonic acid, an inhibitor of phenylpropanoid pathway, increased B. cinerea disease symptoms. In addition, we show that B. cinerea induced expression of genes associated with SAR, general stress and ethylene/jasmonate-mediated defense pathways. Thus, B. cinerea does not induce SAR nor is it affected by SAR, making it a rare example of a necrogenic pathogen that does not cause SAR.     

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P₀. Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P₀ and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK _c 1) gene was increased specifically in the incompatible interaction with TMV-P₀. The expression of CaPK _c 1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK _c 1 responds to several defense-related abiotic stresses in addition to TMV infection. The nucleotide sequence data reported in this paper were submitted to the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number DQ114474.