Abstract
Root-knot nematode, Meloidogyne incognita, is a devastating sedentary endoparasite that causes considerable damage to agricultural crops worldwide. Modern approaches targeting the physiological processes have confirmed the potential of FMRFamide like peptide (FLPs) family of neuromotor genes for nematode management. Here, we assessed the knock down effect of Mi-flp1, Mi-flp12, and Mi-flp18 of M. incognita and their combinatorial fusion cassette on infection and reproduction. Comparative developmental profiling revealed higher expression of all three FLPs in the infective 2nd stage juveniles (J2s). Further, Mi-flp1 expression in J2s could be localized in the ventral pharyngeal nerves near to metacarpal bulb of the central nervous system. In vitro RNAi silencing of three FLPs and their fusion cassette in M. incognita J2s showed that combinatorial silencing is the most effective and affected nematode host recognition followed by reduced penetration ability and subsequent infection into tomato and adzuki bean roots. Northern blot analysis of J2s soaked in fusion dsRNA revealed the presence of siRNA of all three target FLPs establishing successful processing of fusion gene dsRNA in the J2s. Further, evaluation of the fusion gene cassette is done through host-delivered RNAi in tobacco. Transgenic plants with fusion gene RNA-expressing vector were generated in which transgene integration was confirmed by PCR, qRT-PCR, and Southern blot analysis. Transcript accumulation of three FLPs constituting the fusion gene was reduced in the M. incognita females collected from the transgenic plants that provided additional evidence for successful gene silencing. Evaluation of positive T1 transgenic lines against M. incognita brought down the disease burden as indicated by various disease parameters that ultimately reduced the nematode multiplication factor (MF) by 85% compared to the wild-type plants. The study establishes the possibility of simultaneous silencing of more than one FLPs gene for effective management of M. incognita.
Highlights
Plant-parasitic nematodes (PPNs) have proven to be one of the most challenging to control and stubborn pests causing an estimated yield loss amounting to $US 173 billion globally (Elling, 2013)
Target gene silencing through soaking in Double-stranded RNAs (dsRNAs) of three FMRFamide-like peptides (FLPs) genes singly and fusion gene resulted in reduction in average galling by 54% compared to the control
The expression of Mi-flp1, Mi-flp12, and Miflp18 in M. incognita females was down-regulated significantly (P < 0.05) by 0.7 ± 0.2 to 1.5 ± 0.1 fold in case of Mi-flp1, 0.9 ± 0.1 to 1.8 ± 0.2 fold in case of Mi-flp12, and 1.2 ± 0.2 to 2.2 ± 0.3 fold in case of Mi-flp18, respectively, compared to females from WT plants (Figure 9)
Summary
Plant-parasitic nematodes (PPNs) have proven to be one of the most challenging to control and stubborn pests causing an estimated yield loss amounting to $US 173 billion globally (Elling, 2013). Multiple Sequence alignment of these known FLPs in RKNs exhibited less sequence similarity among them despite they share a typical RF-amide sequence at C-terminus Their uniqueness could be advantageous at generating sequence specific knockdown module by dsRNA approach to avoid any off target effects. Increasing evidence for the development of RNAi based transgenics suggests that expression of M. incognita specific dsRNA molecule in the host plant may provide alternative management strategy. Chan et al (2015) delivered a dual gene construct, which included CeCPI and a PjCHI-1 genes in transgenic tomato plants with synthetic promoter, pMSPOA, having NOS-like and SP8a elements. The present investigation strengthens our knowledge on the effect of M. incognita Mi-flp, Mi-flp, Mi-flp and their fusion gene on nematode reproduction and plant parasitism potential when applied through host-delivered RNAi
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