Insights on Novel Effectors and Characterization of Metacaspase (RS107_6) as a Potential Cell Death-Inducing Protein in Rhizoctonia solani.

Effectors play an important role in host-pathogen interactions. Though an economically significant disease in rice, knowledge regarding the infection strategy of Rhizoctonia solani is obscure. In this study, we performed a genome-wide identification of the effectors in R. solani based on the characteristics of previously reported effector proteins. A total of seven novel effectors (designated as RS107_1 to RS107_7) in the disease mechanism of R. solani were identified and were predicted to be non-classically secreted proteins with functionally conserved domains. The function, reactivity, and stability of these proteins were evaluated through physiochemical characterization. The target proteins involved in the regulation of rice defense mechanisms were identified. Furthermore, the effector genes were cloned and RS107_6 (metacaspase) was heterologously expressed in Escherichia coli to obtain a purified protein of ~36.5 kDa. The MALD-TOF characterization confirmed that the protein belonged to a metacaspase of the Peptidase_C14 protein family, 906 bp in size, and encoded a polypeptide of 301 amino acids. These findings suggest that the identified effectors can potentially serve as a virulence factor and can be targeted for the management of sheath blight in rice.

Meloidogyne incognita (Nematoda: Meloidogynidae) sterol-binding protein Mi-SBP-1 as a target for its management.

Meloidogyne incognita is a polyphagous plant-parasitic nematode that causes considerable yield loss in agricultural and horticultural crops. The management options available for M. incognita are extremely limited. Here we identified and characterised a M. incognita homolog of Caenorhabditis elegans sterol-binding protein (Mi-SBP-1), a transcriptional regulator of several lipogenesis pathway genes, and used RNA interference-mediated gene silencing to establish its utility as a target for the management of M. incognita. Mi-sbp-1 is predicted to be a helix-loop-helix domain containing DNA binding transcription factor, and is present in the M. incognita genome in three copies. The RNA-Seq analysis of Mi-sbp-1 silenced second stage juveniles confirmed the key role of this gene in lipogenesis regulation in M. incognita. In vitro and host-induced gene silencing of Mi-sbp-1 in M. incognita second stage juveniles resulted in loss of nematodes' ability to utilise the stored fat reserves, slower nematode development, and reduced parasitism on adzuki bean and tobacco plants. The multiplication factor for the Mi-sbp-1 silenced nematodes on adzuki bean plants was reduced by 51% compared with the control nematodes in which Mi-sbp-1 was not silenced. Transgenic expression of the double-stranded RNA construct of the Mi-sbp-1 gene in tobacco plants caused 40-45% reduction in M. incognita multiplication, 30-43.8% reduction in the number of egg masses, and 33-54% reduction in the number of eggs per egg mass compared with the wild type control plants. Our results confirm that Mi-sbp-1 is a key regulator of lipogenesis in M. incognita and suggest that it can be used as an effective target for its management. The findings of this study can be extended to develop methods to manage other economically important parasitic nematodes.

Knockdown of a mucin-like gene in Meloidogyne incognita (Nematoda) decreases attachment of endospores of Pasteuria penetrans to the infective juveniles and reduces nematode fecundity.

Mucins are highly glycosylated polypeptides involved in many host-parasite interactions, but their function in plant-parasitic nematodes is still unknown. In this study, a mucin-like gene was cloned from Meloidogyne incognita (Mi-muc-1, 1125 bp) and characterized. The protein was found to be rich in serine and threonine with numerous O-glycosylation sites in the sequence. Quantitative real-time polymerase chain reaction (qRT-PCR) showed the highest expression in the adult female and in situ hybridization revealed the localization of Mi-muc-1 mRNA expression in the tail area in the region of the phasmid. Knockdown of Mi-muc-1 revealed a dual role: (1) immunologically, there was a significant decrease in attachment of Pasteuria penetrans endospores and a reduction in binding assays with human red blood cells (RBCs), suggesting that Mi-MUC-1 is a glycoprotein present on the surface coat of infective second-stage juveniles (J2s) and is involved in cellular adhesion to the cuticle of infective J2s; pretreatment of J2s with different carbohydrates indicated that the RBCs bind to J2 cuticle receptors different from those involved in the interaction of Pasteuria endospores with Mi-MUC-1; (2) the long-term effect of RNA interference (RNAi)-mediated knockdown of Mi-muc-1 led to a significant reduction in nematode fecundity, suggesting a possible function for this mucin as a mediator in the interaction between the nematode and the host plant.

A novel in vitro chemotaxis bioassay to assess the response of Meloidogyne incognita towards various test compounds.

Plant-parasitic, root-knot nematodes (Meloidogyne spp.) are a serious problem in agri- and horticultural crops worldwide. Understanding their complex host recognition process is essential for devising efficient and environmental-friendly management tactics. In this study, the authors report a new, simple, inexpensive, efficient, and quantitative method to analyze the chemotaxis of M. incognita second-stage juveniles (J2s) using a combination of pluronic gel and agar in a petri dish. The authors quantitatively defined the concentration gradient formation of acid fuchsin on the assay plate. Using this novel assay method, the authors have accurately measured the nematode response (attraction or repulsion) to various volatile (isoamyl alcohol, 1-butanol, benzaldehyde, 2-butanone, and 1-octanol) and non-volatile (root exudates of tomato, tobacco, and marigold) compounds. Isoamyl alcohol, 1-butanol, and 2-butanone were attractive to J2s through a broad range of concentrations. On the contrary, J2s were repelled when exposed to various concentrations of 1-octanol. Despite being attractive at lower concentrations, undiluted benzaldehyde was repulsive to J2s. Tomato and tobacco root exudates were attractive to J2s while marigold root exudates repelled J2s. The present quantitative assay method could be used as a reference to screen and identify new candidate molecules that attract or repel nematodes.Plant-parasitic, root-knot nematodes (Meloidogyne spp.) are a serious problem in agri- and horticultural crops worldwide. Understanding their complex host recognition process is essential for devising efficient and environmental-friendly management tactics. In this study, the authors report a new, simple, inexpensive, efficient, and quantitative method to analyze the chemotaxis of M. incognita second-stage juveniles (J2s) using a combination of pluronic gel and agar in a petri dish. The authors quantitatively defined the concentration gradient formation of acid fuchsin on the assay plate. Using this novel assay method, the authors have accurately measured the nematode response (attraction or repulsion) to various volatile (isoamyl alcohol, 1-butanol, benzaldehyde, 2-butanone, and 1-octanol) and non-volatile (root exudates of tomato, tobacco, and marigold) compounds. Isoamyl alcohol, 1-butanol, and 2-butanone were attractive to J2s through a broad range of concentrations. On the contrary, J2s were repelled when exposed to various concentrations of 1-octanol. Despite being attractive at lower concentrations, undiluted benzaldehyde was repulsive to J2s. Tomato and tobacco root exudates were attractive to J2s while marigold root exudates repelled J2s. The present quantitative assay method could be used as a reference to screen and identify new candidate molecules that attract or repel nematodes.