RNAi targeting the putative secreted effector candidate Minc09978 and its paralogues impairs host invasion and reproductive fitness in Meloidogyne incognita

Root-knot nematodes ( Meloidogyne spp.) are obligate plant parasites that secrete effector proteins to manipulate host physiology and establish feeding sites. In this study, we investigated Minc09978 , a differentially expressed Meloidogyne incognita gene identified through dual host-nematode transcriptome profiling of infected tomato roots and subsequently shortlisted as a putative secreted effector candidate through computational screening. In silico analysis of the Minc09978 protein identified a predicted N-terminal signal peptide, no detectable transmembrane helices, and a high EffectorP prediction score. Phylogenetic and sequence analyses identified three closely related paralogues in M. incognita and candidate homologues in a subset of the surveyed Meloidogyne species, indicating conservation together with species-associated sequence diversification. Developmental expression profiling revealed transient enrichment of Minc09978 transcripts during the invading J2 stage at 16 h post-inoculation, coinciding with host invasion. RNAi targeting Minc09978 also reduced transcript abundance of its closely related paralogues and significantly impaired nematode attraction, root penetration, and reproduction on tomato and adzuki bean. TRV-mediated transient host-delivered RNAi in tomato reduced Minc09978 transcript abundance in feeding nematodes by 28% and decreased the nematode multiplication factor by 65.9%. Similarly, stable host-delivered RNAi in transgenic Arabidopsis thaliana , using a hairpin construct targeting a conserved region shared by Minc09978 and its paralogues, reduced Minc09978 transcript abundance by up to 71% and the nematode multiplication factor by up to 71.9%. Small-RNA sequencing independently confirmed pronounced accumulation of target-region-derived small RNAs in the high-expressing HD-RNAi line E2, with markedly lower abundance in the low-expressing line E7 and no corresponding reads detected in wild-type plants. qRT-PCR analysis of selected host defence- and secondary-metabolism associated genes in an infected HD-RNAi line revealed reduced expression of several defence-associated markers together with induction of stress- and defence-regulatory genes and a prominent flavonoid/anthocyanin-associated gene set relative to infected wild-type plants. These exploratory expression data indicate that family-level RNAi is associated with changes in selected host defence- and secondary-metabolism-related transcripts during nematode infection. Collectively, the results demonstrate that RNAi-mediated suppression of Minc09978 and its closely related paralogues compromises host invasion and reproductive fitness of M. incognita . The findings support the classification of the Minc09978 protein as a putative secreted effector candidate and identify the Minc09978 paralogue family as a potential target for RNAi-based root-knot nematode management.