Molecular Characterization and Virus-Induced Gene Silencing of a Collagen Gene, Me-col-1, in Root-Knot Nematode Meloidogyne enterolobii

Abstract

Meloidogyne enterolobii, a highly pathogenic root-knot nematode species, causes serious damage to agricultural production worldwide. Collagen is an important part of the nematode epidermis, which is crucial for nematode shape maintenance, motility, and reproduction. In this study, we report that a novel collagen gene, Me-col-1, from the highly pathogenic root-knot nematode species Meloidogyne enterolobi was required for the egg formation of this pathogen. Me-col-1 encodes a protein with the size of 35 kDa, which is closely related to collagen found in other nematodes. Real-time PCR assays showed that the expression of Me-col-1 was highest in eggs and lowest in pre-parasitic second-stage juveniles (preJ2). Interestingly, knockdown of Me-col-1 did not compromise the survival rate of preJ2 but significantly reduced the egg production and consequentially caused 35.79% lower multiplication rate (Pf/Pi) compared with control. Our study provides valuable information for better understanding the function of collagen genes in the nematode life cycle, which can be used in the development of effective approaches for nematode control.

Keywords: Meloidogyne enterolobii; RNAi; VIGS; collagens; root-knot nematode.

Figure 1

Sequence characterization of Me-col-1 gene. (a) Multiple sequence alignment of Me-col-1 with collagens of other nematodes. Shading characters indicate identical (black) or similar (grey) amino acids. The transmembrane region sequence of all Cols is denoted with dotted lines. Nematode cuticle collagen N-terminal domain is marked with overbar. The conserved collagen triple helix repeat domain is underlined. Conserved cysteine residues are marked with rhombuses. (b) Conserved pattern of Me-col-1 cysteine residues. The numbers in parentheses represent the number of Gly-X-Y repeats.

Figure 2

Phylogenetic analysis of Me-col-1 (underline) with collagens of other nematodes. RKN, root-knot nematode; PPN, plant-parasitic nematode; FLN, free-living nematode; APN, animal-parasitic nematode.

Figure 3

SDS-PAGE analysis of Me-col-1 fusion protein. Lane 1, marker; Lane 2–5, Me-col-1 fusion protein induced by IPTG at final concentrations of 0.2 mM, 0.4 Mm, 0.8 mM, 1.0 mM, respectively; Lane 6, pET-32a.

Figure 4

(a) The characterization of nematodes at preJ2, parJ2, J3, J4, female, and egg life stages. The length of the ruler is 20 um; (b) Expression level analysis of Me-col-1 at different developmental stages of M. enterolobii, including preJ2, parJ2, J3/J4, female, and egg life stages. Each bar represents the mean of RT-qPCR reactions run in triplicate, with use of standard error. Significant differences between values were derived by Tukey’s Test (** p ≤ 0.01). Three independent experiments were performed with similar results.

Figure 5

(a) Electrophoretic results of Me-col-1 dsRNA after purification and recovery. M, marker; (b) Death rate of M. enterolobii after in vitro RNAi.

Figure 6

Effect of in planta RNAi of Me-col-1. (a) The TRV cp gene in N. benthamiana roots was detected by PCR 7 days after TRV inoculation. M: marker, Col: TRV2-Col, EX: empty vector control TRV2-EX; (b) the numbers of galls in the N. benthamiana roots; (c) average number of eggs per plant; (d) the multiplication rate (Pf/Pi) of TRV inoculated plants. Data are presented as means from 10 plants. The independent experiments were repeated twice with similar results. (** p ≤ 0.01).