实时荧光RT-PCR检测辣椒轻斑驳病毒的初步研究

本研究选取辣椒轻斑驳病毒PMMoV、烟草花叶病毒TMV、黄瓜花叶病毒CMV和马铃薯Y病毒PVY作为试验材料,根据PMMoV衣壳蛋白(CP)RNA的序列特异性位点,设计出Taqman荧光探针及其引物,采用实时荧光RT-PCR技术对PMMoV进行快速检测,同时与ELISA方法进行了灵敏度比较,结果表明：该方法具有较高的灵敏度及较强的特异性,PMMoV检测结果为阳性,TMV、CMV和PVY均无荧光信号,为阴性,灵敏度是传统的ELISA方法的100倍,而且大大缩短了检测时间。该方法快速、准确、灵敏、简便、安全,具有实际应用价值,适用于植物病毒病害的快速检测。     

Immunolocalization of Pepper mild mottle virus in Capsicum annuum seeds

To clarify the mechanism of seed transmission of Pepper mild mottle virus (PMMoV), the virus was immunolocalized in Capsicum annuum seeds using fluorescence microscopy. Two distinct patterns were observed: In the first, PMMoV was present in the epidermis and parenchyma but not in the endosperm or embryo; in the second, the virus was restricted to the surface of the epidermis and parenchyma. These findings shed light on the fundamental mechanisms of seed transmission of tobamoviruses and may aid in the design of new methods to prevent the spread of seedborne virus diseases.     

侵染四川辣椒的番茄斑萎病毒和辣椒轻斑驳病毒的小RNA测序检测及鉴定

 调查发现四川省汶川县当地辣椒的病毒病严重且症状多样,病样粗提液摩擦接种辣椒、矮牵牛和三生烟,出现辣椒系统性花叶焦枯和茎尖坏死,指示植物表现局部枯斑。对3个不同症状的病果进行sRNA深度测序鉴定,发现均含有番茄斑萎病毒(Tomato spotted wilt virus,TSWV)和辣椒轻斑驳病毒(Pepper mild mottle virus,PMMoV)。通过RT-PCR技术进行验证,结果显示所有病样的果皮和部分新鲜种子以及回接寄主的病叶均检测到TSWV和PMMoV,表明该地辣椒病毒病是由TSWV和PMMoV复合侵染引起。这是TSWV侵染四川辣椒的首次报道。分别基于TSWV N基因序列和PMMoV CP基因序列构建系统发育树,汶川分离物TSWV-WC(MK468469)与贵州分离物(KP684518)亲缘关系最近,PMMoV-WC(MK408614)与北京分离物(AY859497)亲缘关系最近。推测该地辣椒病毒病可能与品种引进有关。     

Timing and extent of hypersensitive response are critical to restrict local and systemic spread of Pepper mild mottle virus in pepper containing the L 3 gene

Amino acid changes in Pepper mild mottle virus (PMMoV) coat protein (CP) that enhance, decrease, or nullify the resistance-inducing activity in Capsicum plants carrying the L ³ gene have been identified. In this study, molecular events underlying the L ³ -gene-mediated resistance were analyzed through the expression of hypersensitive response (HR)-related genes, HSR203J-Cc and HIN1-Cc, and defense-related genes, PR1-Cc and PR4b-Cc, upon infection with PMMoV CP mutants. The expression kinetics of the genes correlated with the degree of restriction of virus distribution in the inoculated leaves. The results suggest that the timing and extent of HR are critical factors to restrict virus spread both locally and systemically in L ³ -gene-mediated resistance.The nucleotide sequence data reported are available in the DDBJ/GenBank/EMBL databases under accession numbers AB162220 (HSR203J-Cc), AB162221 (HIN1-Cc), AB162222 (PR1-Cc), and AB162223 (PR4b-Cc)     

Two Amino Acid Substitutions in the Coat Protein of Pepper mild mottle virus Are Responsible for Overcoming the L(4) Gene-Mediated Resistance in Capsicum spp

ABSTRACT The Capsicum spp. L genes (L(1) to L(4)) confer resistance to tobamoviruses. Currently, the L(4) gene from Capsicum chacoense is the most effective resistance gene and has been used widely in breeding programs in Japan which have developed new resistant cultivars against Pepper mild mottle virus (PMMoV). However, in 2004, mild mosaic symptoms began appearing on the leaves of commercial pepper plants in the field which possessed the L(4) resistance gene. Serological and biological assays on Capsicum spp. identified the causal virus strain as a previously unreported pathotype, P(1,2,3,4). PMMoV sequence analysis of the virus and site-directed mutagenesis using a PMMoV-J of the P(1,2) pathotype revealed that two amino acid substitutions in the coat protein, Gln to Arg at position 46 and Gly to Lys at position 85, were responsible for overcoming the L(4) resistance gene.     

Amino Acid 129 of Cucumber mosaic virus Coat Protein Determines Local Symptom Expression and Systemic Movement in Tetragonia expansa, Momordica charantia and Physalis floridana

Local symptom expression and systemic movement of Cucumber mosaic virus (CMV) in Tetragonia expansa, Momordica charantia and Physalis floridana were mapped to the amino acid at position 129 of CMV coat protein (CP), using pseudorecombinants, chimeric RNAs, a site-directed mutant of RNA 3 and four strains of CMV : pepo-, SO-, MY17- and Y-CMV. Local and systemic symptoms caused by three strains, pepo-, SO- and MY17-CMV, and those by Y-CMV differed in the three host species. The three strains expressed local chlorotic spots at 24°C and systemic chlorotic spots and ringspots at 36°C, whereas Y-CMV developed local necrotic spots at 24°C but no systemic symptoms at 36°C in T. expansa. In M. charantia the three strains caused systemic chlorotic spots, whereas Y-CMV caused local necrotic spots. The three caused systemic mosaic and Y-CMV systemic necrosis in P. floridana. With pseudorecombinants combined with pepo- and Y-CMV RNAs, CMV RNA 3 was responsible for symptom expression and systemic infection. Inoculation with Y-CMV RNA 1, RNA 2 and chimeric RNA 3s exchanged CP gene fragments between pepo- and Y-CMV showed that NruI-XhoI fragment of CP was essential for symptom expression. Comparative analysis of the NruI-XhoI fragments revealed that only the amino acid at position 129 was common among the three strains but different from that of Y-CMV. Inoculation with a point mutant constructed by substituting one nucleotide resulting in an amino acid change from Ser to Pro at position 129 in Y-CMV CP verified the previous experiments. These results indicate that the amino acid at position 129 of CMV CP is the determinant for local symptom expression and systemic movement in the three host species. CMV CP containing Ser at position 129 may induce resistant responses in these plants. Received 29 June 2001/ Accepted in revised form 28 August 2001     

Pepper 9- and 13-lipoxygenase genes are differentially activated by two tobamoviruses and by hormone treatments

Two novel pepper 13-lipoxygenase (LOX) genes were cloned and their expressions were compared with those of three 9-LOX genes in pepper leaves inoculated with two different tobamoviruses. Obuda pepper virus (ObPV) inoculation led to a massive induction of pathogenesis-related genes and to the development of hypersensitive reaction (incompatible interaction), while Pepper mild mottle virus (PMMoV) inoculation resulted in a compatible interaction. Both virus infections markedly activated the expression of the two novel 13-LOXs. The magnitudes of induction of 13-LOXs did not differ substantially between the ObPV- and PMMoV-inoculated leaves. The induction of three 9-LOXs was markedly more robust and rapid in ObPV-inoculated leaves than in PMMoV-inoculated ones. LOXs were very differentially activated in pepper leaves treated with defense hormones. A large number of hormone-related cis-regulatory elements were identified in the promoter regions of LOXs. ObPV inoculation resulted also in the substantial up-regulation of an omega-6-fatty acid desaturase gene. Our results suggest that 9-LOX-dependent pathways are more probably involved in the suppression of virus replication than 13-LOX-dependent plant responses.