RNAi-induced silencing of an effector confers transcriptional oscillation in another group of effectors in the root-knot nematode, Meloidogyne incognita

Abstract

The sophisticated parasitic tactic of sedentary endoparasitic nematodes seems to involve the simultaneous alteration of the expression of multitude of its effector genes in order to hijack the plant metabolic and developmental pathway. In concordance with this hypothesis, we have targeted some candidate effector genes of Meloidogyne incognita to understand the possible interaction among those effectors for successful infection of the host plant. In vitro RNAi strategy was used to knock down M. incognita-specific pioneer effector genes, such as msp-18, msp-20, msp-24, msp-33 and msp-16 (known to interact with plant transcription factor), to investigate their possible effect on the expression of key cell wall-degrading enzymes (CWDE) and vice versa. Supported by the phenotypic data, intriguingly our study revealed that induced suppression of these pioneer genes cause transcriptional alteration of CWDE genes in M. incognita. This remarkable finding may provide some useful links for future research on nematode effector interaction.

Host-Induced Silencing of Two Pharyngeal Gland Genes Conferred Transcriptional Alteration of Cell Wall-Modifying Enzymes of Meloidogyne incognita vis-à-vis Perturbed Nematode Infectivity in Eggplant

“…Total RNA (400 ng) was isolated from the pre-parasitic J2 of M. incognita , their quality and quantity were assessed and converted to cDNA as described by Shivakumara et al (2016). Fragments of five CWMEs ( Mi-xyl-1 : AF224342, Mi-xyl-3 : EU475876, Mi-pg-1 : AY098646, Mi-eng-1 : AF100549, and Mi-pel : AF527788) and two pioneer genes ( msp-18 : AY134437 and msp-20 : AY134439) were PCR amplified, cloned into pGEM-T vector and identity of the insert was confirmed via sequencing as described in Shivakumara et al (2016).…”

“…Fragments of five CWMEs ( Mi-xyl-1 : AF224342, Mi-xyl-3 : EU475876, Mi-pg-1 : AY098646, Mi-eng-1 : AF100549, and Mi-pel : AF527788) and two pioneer genes ( msp-18 : AY134437 and msp-20 : AY134439) were PCR amplified, cloned into pGEM-T vector and identity of the insert was confirmed via sequencing as described in Shivakumara et al (2016). Primer details are documented in Shivakumara et al (2016).…”

“…RNA was extracted from different stages of M. incognita and reverse transcribed to cDNA as described above. All qRT-PCR primers (Shivakumara et al, 2016) were designed using IDT OligoAnalyzer 3.1 software 1 and optimized for working concentration and annealing temperature prior to use with a Realplex 2 thermal cycler instrument (Eppendorf). The 10 μl reaction mixture contained 1.5 ng of cDNA, 750 nM of each primer and 5 μl of SYBR Green PCR master-mix (Eurogentec).…”

“…It is assumed that the concurrent expression of several effectors is required in order to successfully parasitize the host plants by nematodes (Mitchum et al, 2013), although to support this notion enough experimental proofs are not yet available. Recently, using in vitro and in vivo reverse-genetics approach we have demonstrated (Shivakumara et al, 2016) the existence of crosstalk among various Meloidogyne incognita effectors the origin of which encompass both the subventral (CWMEs – xylanases: Mi-xyl-1 , Mi-xyl-3 , β-1,4-endoglucanase: Mi-eng-1 , pectate lyase: Mi-pel , polygalacturonase: Mi-pg-1 , Haegeman et al, 2012; pioneer gene – msp-20 , Huang et al, 2003) and dorsal esophageal glands (pioneer genes: msp-16 , msp-18 , msp-24 , msp-33 ; Huang et al, 2003). It was shown that the induced suppression of one effector gene (subventral gland-specific) leads to the transcriptional alteration of other unrelated effectors (dorsal gland-specific) and vice versa in pre-parasitic J2s of M. incognita .…”

“…It was shown that the induced suppression of one effector gene (subventral gland-specific) leads to the transcriptional alteration of other unrelated effectors (dorsal gland-specific) and vice versa in pre-parasitic J2s of M. incognita . In addition, knockdown of pioneer genes (supposedly play minimal role in nematode migration) led to the reduced penetration ability of M. incognita in tomato (Shivakumara et al, 2016). These findings suggested that there is a potential link between the expression of subventral gland-specific CWMEs and dorsal gland-specific pioneer genes during early stage of nematode infection process.…”