Knocking-down Meloidogyne incognita proteases by plant-delivered dsRNA has negative pleiotropic effect on nematode vigor

Abstract

The root-knot nematode Meloidogyne incognita causes serious damage and yield losses in numerous important crops worldwide. Analysis of the M. incognita genome revealed a vast number of proteases belonging to five different catalytic classes. Several reports indicate that M. incognita proteases could play important roles in nematode parasitism, besides their function in ordinary digestion of giant cell contents for feeding. The precise roles of these proteins during parasitism however are still unknown, making them interesting targets for gene silencing to address protein function. In this study we have knocked-down an aspartic (Mi-asp-1), a serine (Mi-ser-1) and a cysteine protease (Mi-cpl-1) by RNAi interference to get an insight into the function of these enzymes during a host/nematode interaction. Tobacco lines expressing dsRNA for Mi-ser-1 (dsSER), Mi-cpl-1 (dsCPL) and for the three genes together (dsFusion) were generated. Histological analysis of galls did not show clear differences in giant cell morphology. Interestingly, nematodes that infected plants expressing dsRNA for proteases produced a reduced number of eggs. In addition, nematode progeny matured in dsSER plants had reduced success in egg hatching, while progeny resulting from dsCPL and dsFusion plants were less successful to infect wild-type host plants. Quantitative PCR analysis confirmed a reduction in transcripts for Mi-cpl-1 and Mi-ser-1 proteases. Our results indicate that these proteases are possibly involved in different processes throughout nematode development, like nutrition, reproduction and embryogenesis. A better understanding of nematode proteases and their possible role during a plant-nematode interaction might help to develop new tools for phytonematode control.

Figure 1. In silico analyses of all Meloidogyne incognita aspartic, serine and cysteine proteases ESTs present in EST data bank dbEST.

Representation of M. incognita proteases expressed sequence tags (ESTs) in databanks. Bars show the percentage of proteases EST number relative to the total number of EST available for each developmental stage. ESTs from proteases were retrieved from NCBI-dbEST (http://www.ncbi.nlm.nih.gov/dbEST/index.html) and their representation was assessed by the number of ESTs relative to the total number of ESTs available for the developmental stage considered. The developmental stages considered were; eggs (14,671 ESTs), freshly hatched J2s (33,835 ESTs), mixed parasitic stages (3,133 ESTs) and females (4,427 ESTs). The distribution of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ESTs is indicated for comparison.

Figure 2. Relative abundance of specific protease gene transcripts in Meloidogyne incognita.

Real-time qRT-PCR analysis of M. incognita proteases transcript levels at different stages of the nematode life cycle. (A) Cathepsin D-like aspartic proteinase (Mi-asp-1, Accession: DQ360827). (B) Chymotrypsin-like serine proteinase (Mi-ser-1, AY714229). (C) Cathepsin L cystein proteinase (Mi-cpl-1, AJ557572). Each bar represents the mean of duplicate assays repeated twice. Standard errors are shown. Different letters mean statistical difference (p≤0.05) according to the iteration test (Rest 2009 Software). The results are presented as fold change in comparison to the stage that had the smaller relative expression value that was arbitrarily designed as 1.

Figure 3. Gene cloning and transgenic tobacco plant generation for host-derived RNA-interference of Meloidogyne incognita proteases.

(A) Regions of proteinases genes used in RNAi experiments. Numbers indicate nucleotide positions. (B) Schematic representation of the pK7GWIWG2(I) (Karimi et al. 2002) hairpin double-stranded RNA (dsRNA) constructs containing the sense and antisense coding regions fragments of Mi-asp-1, Mi-ser-1, Mi-cpl-1 separately and together. (C) Characterization of RNAi lines for silencing of Mi-ser-1, Mi-cpl-1 and the fragments in tandem of Mi-asp-1, Mi-ser-1 and Mi-cpl-1 (Fusion), by PCR. Attempts for generate ds-Mi-asp-1 lines were not successful. Sense (S) fragment, anti-sense (AS) fragment, undistinguishable fragment (Sense or Anti-sense) (F). (D) RT-PCR of the single-stranded pK7GWIWG2(I) intron (spacer) of the hairpin dsRNA was used to confirm the expression of Mi-ser-1, Mi-cpl-1 and fusion dsRNAs in seedlings of independent transgenic tobacco lines at 15 d post-germination.

Figure 4. Meloidogyne incognita morphology (biometry) 28 days after inoculation (DAI) that matured on transgenic plants expressing dsRNA nematode proteases.

Plot showing the size and morphology of Meloidogyne incognita at 28 DAI. The length and roundness are indicated by the vertical and horizontal axis, respectively. This plot allows assignment of individual nematodes within two classes: young female (lower quadrant) and mature female (upper quadrant) according to Atkinson et al. [40]. Representative fuchsin stained nematodes are also shown (bar = 100 µM) illustrating examples in shape and size of the two classes.

Figure 5. Knock-down of Meloidogyne incognita proteases affects nematode reproduction and egg viability (after 45 DAI).

(A) Number of eggs per gram of root. (B) Total egg hatching ratio. Experiments were repeated twice. Different letters mean statistical significance through one-way ANOVA and Tukey test (p≤0.05).

Figure 6. Quantitative RT-PCR showing the transcript levels of proteases in eggs exposed to dsRNAs.

Eggs were collected from M. incognita females that infected transgenic tobacco lines expressing dsRNA for Mi-SER, Mi-CPL and Mi-ASP, Mi-SER and Mi-CPL fused (fusion). (A) Analysis of Mi-asp-1; in M. incognita (Mi) eggs of 35S-dsCPL and 35S-dsSER plants Mi-asp-1 was not exposed to a specific dsRNA. (B) Analysis of Mi-cpl-1 gene; in Mi eggs of 35S-dsSER plants Mi-cpl-1 was not exposed to a specific dsRNA. (C) Analysis of Mi-ser-1 gene; in Mi eggs from 35S-dsCPL plants Mi-ser-1 was not exposed to a specific dsRNA. Significant differences were assessed by Iteration test (REST Software) where proteases gene expression in nematodes eggs from different plants lines were compared to control plants (*, **, *** = P ≤ 0.05, 0.01 and 0.001, respectively).

Figure 7. Effect of protease knock-down on progeny virulence of M. incognita.

Hatched J2 from eggs that were laid by females that feed on transgenic and control plants were inoculated in wild-type tobacco. (A) Relative number of galls per plant at 45 DAI; (B) Relative number of egg masses per plant at 45 DAI; Experiments were repeated twice. Different letters mean statistical significance through one-way ANOVA and Tukey test (P≤0.05).