Meloidogyne incognita is a devastating plant-parasitic nematode.  Effectors play important roles during the stages of nematodes infection and parasitism, but their molecular functions remain largely unknown.  In this study, we characterized a new effector, Minc03329, which contains signal peptide for secretion and a C-type lectin domain.  The yeast signal sequence trap experiments indicated that the signal peptide of Minc03329 is functional.  In situ hybridization showed that Minc03329 was specifically expressed in the subventral esophageal gland.  Real-time qPCR confirmed that the expression level of Minc03329 transcript was significantly increased in pre-parasitic and parasitic second-stage juveniles (pre-J2s and par-J2s).  Tobacco rattle virus (TRV)-mediated gene silencing of Minc03329 in host plants largely reduced the pathogenicity of nematodes.  On the contrary, ectopic expression of Minc03329 in Arabidopsis thaliana significantly increased plant susceptibility to nematodes.  Transient expression of Minc03329 in Nicotiana benthamiana leaves suppressed the programmed cell death triggered by the pro-apoptotic protein BAX.  Moreover, the transcriptome analysis of Minc03329-transgenic Arabidopsis and wild type revealed that many defense-related genes were significantly down-regulated.  Interestingly, some different expressed genes were involved in the formation of nematode feeding sites.  These results revealed that Minc03329 is an important effector for M. incognita, suppressing host defense response and promoting pathogenicity.

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.

   Cucumber mosaic virus (CMV) can infect a wide range of host species. For the lacking of CMV resistant varieties of tomato, RNA interference (RNAi) can be used as a fast and effective method for the generation of transgenic resistant varieties. In this current study, five intron-spliced hairpin RNA (ihpRNA) plant expression vectors aimed at five genes of CMV have been constructed. Transgenic tomatoes were obtained by Agrobacterium tumefaciens-mediated transformation with expression vectors. Highly resistant generations of transgenic plants were employed as rootstocks and grafted onto non-transgenic tomatoes that resulted in the successful transfer of resistance to the scions. Using a novel method of plant cuttings for rootstock propagation, we obtained large quantities of disease-resistant material. Further, this method produces scions that can remain undetectable for transgenic resistance marker genes that may provide novel approaches to evade collective concerns about genetically-modified organism (GMO) biosafety.

   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.

Fusarium oxysporum f. sp. conglutinans (Foc) is the causal agent of Fusarium wilt disease of Brassica oleracea. A rapid, accurate, and reliable method to detect and identify plant pathogens is vitally important to integrated disease management. In this study, using a comparative genome analysis among Fusarium oxysporum (Fo), we developed a Foc-specific primer set (Focs-1/Focs-2) and established a multiplex-PCR assay. In the assay, the Focs-1/Focs-2 and universal primers for Fusarium species (W106R/F106S) could be used to detect Foc isolates in a single PCR reaction. With the optimized PCR parameters, the multiplex-PCR assay showed a high specificity for detecting Foc and was very sensitive to detect as little as 100 pg of pure Foc genomic DNA or 1 000 spores in 1 g of twice-autoclaved soil. We also demonstrated that Foc isolates were easily detected from infected plant tissues, as well as from natural field soils, using the multiplex-PCR assay. To our knowledge, this is a first report on detection Fo by comparative genomic method.

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.