Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism.

Validation of reference genes aiming accurate normalization of qPCR data in soybean upon nematode parasitism and insect attack.

BACKGROUND: Soybean pathogens and pests reduce grain production worldwide. Biotic interaction cause extensive changes in plant gene expression profile and the data produced by functional genomics studies need validation, usually done by quantitative PCR. Nevertheless, this technique relies on accurate normalization which, in turn, depends upon the proper selection of stable reference genes for each experimental condition. To date, only a few studies were performed to validate reference genes in soybean subjected to biotic stress. Here, we report reference genes validation in soybean during root-knot nematode (Meloidogyne incognita) parasitism and velvetbean caterpillar (Anticarsia gemmatalis) attack. FINDINGS: The expression stability of nine classical reference genes (GmCYP2, GmELF1A, GmELF1B, GmACT11, GmTUB, GmTUA5, GmG6PD, GmUBC2 and GmUBC4) was evaluated using twenty-four experimental samples including different organs, developmental stages, roots infected with M. incognita and leaves attacked by A. gemmatalis. Two different algorithms (geNorm and NormFinder) were used to determine expression stability. GmCYP2 and GmUBC4 are the most stable in different organs. Considering the developmental stages, GmELF1A and GmELF1B genes are the most stable. For spatial and temporal gene expression studies, normalization may be performed using GmUBC4, GmUBC2, GmCYP2 and GmACT11 as reference genes. Our data indicate that both GmELF1A and GmTUA5 are the most stable reference genes for data normalization obtained from soybean roots infected with M. incognita, and GmCYP2 and GmELF1A are the most stable in soybean leaves infested with A. gemmatalis. CONCLUSIONS: Future expression studies using nematode infection and caterpilar infestation in soybean plant may utilize the reference gene sets reported here.

Ectopic expression of a Meloidogyne incognita dorsal gland protein in tobacco accelerates the formation of the nematode feeding site.

Meloidogyne spp., plant-parasitic nematodes present worldwide, are intensively studied because of the damage caused to a large variety of agronomically important crops. Several reports indicate that proteins from the Meloidogyne spp. dorsal gland might play an important role to allow proper establishment of a functional nematode feeding site. The precise role of these proteins in the process of feeding cell development is unknown. To gain insights into the function of these secreted M. incognita proteins, we constitutively (ectopically) expressed the nematodes dorsal gland protein 7E12 in tobacco plants. It was found that the number of galls at 8 and 16 days after nematode infection was significantly higher in transgenic plants compared to control plants. Eggs from nematodes in transgenic plants hatched faster than those in control plants. Histological analysis of nematode induced galls in transgenic plants clearly shows a different morphology. Giant feeding cells harbor more vacuoles and an increased amount of cell wall invaginations, while neighboring cells surrounding feeding cells are more numerous. These results suggest that the presence of the 7E12 protein in tobacco accelerates gall formation. This assumption is supported by our data illustrating faster gall formation and egg eclosion in transgenic plants.