根结线虫病是农业生产上的毁灭性病害，每年造成巨大的经济损失。南方根结线虫是一种寄生范围广、危害严重、防治困难的根结线虫，其防治主要依赖化学农药，不仅污染环境、危害人类健康，而且大大增加线虫的抗药性。解析根结线虫的致害分子机制，对于制定环保、经济、高效的防治策略具有重要研究价值。研究发现，根结线虫在寄生过程由食道腺表达、通过口针分泌出许多效应子，在线虫侵染和寄生阶段发挥重要作用。不同种类效应子与寄主植物之间产生错综复杂的相互作用，功能机制有待深入解析。在本研究中，我们鉴定了一种新的南方根结线虫效应子Minc03329，对氨基酸序列分析发现，其包含用于分泌的信号肽序列和一个C型凝集素结构域。酵母信号序列捕获实验表明Minc03329的信号肽是有功能的，具有分泌功能；原位杂交实验结果表明Minc03329在南方根结线虫亚腹食道腺中特异表达；实时荧光定量PCR结果证实 Minc03329 在线虫寄生初期表达量显著升高；病毒介导的基因沉默干扰线虫 Minc03329 表达，显著降低了南方根结线虫的致病性；相反，Minc03329转基因拟南芥接种南方根结线虫后根结数和卵块数显著增加，表明效应子Minc03329在植物细胞中表达，可以显著增加植物对南方根结线虫的敏感性；Minc03329 在本氏烟草叶片细胞中瞬时表达能抑制由小鼠促凋亡蛋白BAX引发的细胞程序性死亡；通过对Minc03329转基因拟南芥和野生型拟南芥进行转录组数据比较分析，发现Minc03329转基因拟南芥中许多防御相关基因表达显着下调；此外，一些差异表达基因可能参与了南方根结线虫摄食位点的形成，但是其分子机制有待深入解析。本研究是在揭示凝集素效应子MiCTL1功能机制后，解析的第二个南方根结线虫凝集素类效应子的功能。验证了凝集素类效应子在线虫与植物互作过程通过抑制植物免疫反应，帮助线虫寄生的分子机制。研究结果为揭示根结线虫致病分子机理，以及根结线虫防治分子靶标利用提供了重要理论依据。

A continuous co-evolutionary arms-race between pathogens and their host plants promotes the development of pathogenic factors by microbes, including carbohydrate esterase (CE) genes to overcome the barriers in plant cell walls.  Identification of CEs is essential to facilitate their functional and evolutionary investigations; however, current methods may have a limit in detecting some conserved domains, and ignore evolutionary relationships of CEs, as well as do not distinguish CEs from proteases.  Here, candidate CEs were annotated using conserved functional domains, and orthologous gene detection and phylogenetic relationships were used to identify new CEs in 16 oomycete genomes, excluding genes with protease domains.  In our method, 41 new putative CEs were discovered comparing to current methods, including three CE4, 14 CE5, eight CE12, five CE13, and 11 CE14.  We found that significantly more CEs were identified in Phytophthora than in Hyaloperonospora and Pythium, especially CE8, CE12, and CE13 that are putatively involved in pectin degradation.  The abundance of these CEs in Phytophthora may be due to a high frequency of multiple-copy genes, supporting by the phylogenetic distribution of CE13 genes, which showed five units of Phytophthora CE13 gene clusters each displaying a species tree like topology, but without any gene from Hyaloperonospora or Pythium species.  Additionally, diverse proteins associated with products of CE13 genes were identified in Phytophthora strains.  Our analyses provide a highly effective method for CE discovery, complementing current methods, and have the potential to advance our understanding of function and evolution of CEs.

The expression of mitogen-activated protein kinase (mapk) double-stranded RNA in cucumber is effective in controlling infestations of the root-knot nematode Meloidogyne incognita.  However, little is known about the ecological effects of transgenic plants.  Here, we analyzed the diversity of 16S rDNA genes derived from the rhizosphere archaea of transgenic cucumber plants as an indicator of ecological change.  A total of 17 and 18 operational taxonomic units were detected in the rhizospheres of non-transgenic cucumber and mapk dsRNA-expressing plants, respectively.  No significant difference was observed between the two groups according to Shannon and Simpson indices.  In soil samples of the two rhizospheres, the dominant group was Crenarchaeota at the phylum level, with Staphylothermus, Methanococcus, Pyrodictium and Sulfolobus the abundant taxa at the genus level.  These results suggest that expressing mapk double-stranded (ds) RNA in cucumber has no apparent effect on the diversity of rhizosphere archaea, and provide powerful evidence for the ecological safety of transgenic cucumber expressing mapk dsRNA.   

Pepper (Capsicum annuum. L.) is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China.  Pepper root-knot nematode (Meloidogyne spp.) is one of the most serious pests of pepper, which caused huge losses every year.  Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita.  HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chromosome library (BAC) via the vector of CopyControl^TM pCC1 in this study.  The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes.  The average insert size is 95 kb, and most of them are 90–120 kb; but the empty clones are less than 3%.  In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study.  Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained.  Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively.  Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.

   Virus-tolerant plant, which allows the accumulation of virus and then generates virus-derived small RNAs (vsRNAs), is a valuable material to reveal the antiviral efficiency of vsRNAs. Here, a comparison of vsRNAs in Tomato yellow leaf curl virus tolerant and in susceptible tomato varieties showed the consistent trend of vsRNAs’ distribution on virus genome, which is presented as an obvious characteristic. However, the expression level of vsRNA in tolerant variety is less than that in susceptible variety. Slicing targets of vsRNA-mediated viral transcripts were investigated using parallel analysis of RNA ends, and geminivirus DNA methylation was determined by bisulfite sequencing, which uncovered that not all vsRNAs participated in viral mRNA degradation and DNA methylation. Additionally, by comparing with the expression pattern of vsRNAs, viral DNA and mRNA, we proposed the quantity of vsRNAs is corresponding to the expression level of viral mRNA, while the virus-suppression of vsRNAs is not high-efficient.

Root-knot nematodes (RKNs, Meloidogyne spp.) are obligate biotrophic parasites that settle close to the vascular tissues in plant roots. The diseases resulting from RKN infections cause serious damage to agricultural production worldwide. In the present paper, the resistance of Chinese leek (Allium tuberosum Rottler ex Sprengel) against RKNs, its suppressive effect on nematode disease, its nematicidal activity and its component profile were studied to identify an efficient disease control method. In soil heavily infected by nematodes, Chinese leek showed strong resistance to RKNs. Additionally, the gall indexes of cucumber plants rotated with Chinese leek and of tomato plants intercropped with Chinese leek were reduced by 70.2 and 41.1%, respectively. In a pot experiment, the gall indexes of Chinese leek extract-treated tomato and cucumber plants were reduced by 88.9 and 75.9%, respectively. In an in vitro experiment, the mortality rate of a RKN (Meloidogyne incognita J2) treated with Chinese leek extract was significantly higher than that of the control. The gas chromatography- mass spectrometry (GC-MS) analysis revealed that glycosides, carboxylic acids, ketones and organic sulfides are the main components in the Chinese leek extract. This study revealed that Chinese leek possesses a high resistance to RKNs, has strong nematicidal activity against M. incognita and can significantly reduce the incidence of disease caused by nematodes.