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Transcriptomic changes in the pre-parasitic juveniles of Meloidogyne incognita induced by silencing of effectors Mi-msp-1 and Mi-msp-20.

ABSTRACT: Plant-parasitic root-knot nematode Meloidogyne incognita uses an array of effector proteins to establish successful plant infections. Mi-msp-1 and Mi-msp-20 are two known effectors secreted from nematode subventral oesophageal glands; Mi-msp-1 being a putative secretory venom allergen AG5-like protein, whereas Mi-msp-20 is a pioneer gene with a coiled-coil motif. Expression of specific effector is known to cause disturbances in the expression of other effectors. Here, we used RNA-Seq to investigate the pleiotropic effects of silencing Mi-msp-1 and Mi-msp-20. A total of 25.1-51.9 million HQ reads generated from Mi-msp-1 and Mi-msp-20 silenced second-stage juveniles (J2s) along with freshly hatched J2s were mapped to an already annotated M. incognita proteome to understand the impact on various nematode pathways. As compared to control, silencing of Mi-msp-1 caused differential expression of 29 transcripts, while Mi-msp-20 silencing resulted in differential expression of a broader set of 409 transcripts. In the Mi-msp-1 silenced J2s, cytoplasm (GO:0005737) was the most enriched gene ontology (GO) term, whereas in the Mi-msp-20 silenced worms, embryo development (GO:0009792), reproduction (GO:0000003) and nematode larval development (GO:0002119) were the most enriched terms. Limited crosstalk was observed between these two effectors as a sheer 5.9% of the up-regulated transcripts were common between Mi-msp-1 and Mi-msp-20 silenced nematodes. Our results suggest that in addition to the direct knock-down caused by silencing of Mi-msp-1 and Mi-msp-20, the cascading effect on other genes might also be contributing to a reduction in nematode's parasitic abilities.

SUBMITTER: Somvanshi VS

PROVIDER: S-EPMC7391465 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe Mi-1 gene was the first identified and cloned gene that provides resistance to root-knot nematodes (RKNs) in cultivated tomato. However, owing to its temperature sensitivity, this gene does not meet the need for breeding disease-resistant plants that grow under high temperature. In this study, Mi-3 was isolated from the wild species PI 126443 (LA3858) and was shown to display heat-stable resistance to RKNs. However, the mechanism that regulates this resistance remains unknown.ResultsIn this study, 4760, 1024 and 137 differentially expressed genes (DEGs) were enriched on the basis of pairwise comparisons (34 °C vs. 25 °C) at 0 (before inoculation), 3 and 6 days post-inoculation (dpi), respectively. A total of 7035 DEGs were identified from line LA3858 in the respective groups under the different soil temperature treatments. At 3 dpi, most DEGs were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to plant biotic responses, such as "plant-pathogen interaction" and "plant hormone signal transduction". Significantly enriched DEGs were found to encode key proteins such as R proteins and heat-shock proteins (HSPs). Moreover, other DEGs were found to participate in Ca2+ signal transduction; the production of ROS; DEGs encoding transcription factors (TFs) from the bHLH, TGA, ERF, heat-shock transcription factor (HSF) and WRKY families were highly expressed, which contribute to be involved into the formation of phytohormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), the expression of most was upregulated at 3 dpi at the 25 °C soil temperature compared with the 34 °C soil temperature.ConclusionTaken together, the results of our study revealed reliable candidate genes from wild materials LA3858, that are related to Mi-3-mediate resistance to Meloidogyne incognita. A large number of vital pathways and DEGs were expressed specifically in accession LA3858 grown at 34 °C and 25 °C soil temperatures at 3 dpi. Upon infection by RKNs, pattern-recognition receptors (PRRs) specifically recognized conserved pathogen-associated molecular patterns (PAMPs) as a result of pathogen-triggered immunity (PTI), and the downstream defensive signal transduction pathway was likely activated through Ca2+ signal channels. The expression of various TFs was induced to synthesize phytohormones and activate R proteins related to resistance, resulting in the development of effector-triggered immunity (ETI). Last, a hypersensitive response in the roots occurred, which was probably induced by the accumulation of ROS.

Project description:BackgroundSouthern root-knot nematode Meloidogyne incognita (Kofoid and White, 1919), Chitwood, 1949 is a key pest of agricultural crops. Pasteuria penetrans is a hyperparasitic bacterium capable of suppressing the nematode reproduction, and represents a typical coevolved pathogen-hyperparasite system. Attachment of Pasteuria endospores to the cuticle of second-stage nematode juveniles is the first and pivotal step in the bacterial infection. RNA-Seq was used to understand the early transcriptional response of the root-knot nematode at 8 h post Pasteuria endospore attachment.ResultsA total of 52,485 transcripts were assembled from the high quality (HQ) reads, out of which 582 transcripts were found differentially expressed in the Pasteuria endospore encumbered J2 s, of which 229 were up-regulated and 353 were down-regulated. Pasteuria infection caused a suppression of the protein synthesis machinery of the nematode. Several of the differentially expressed transcripts were putatively involved in nematode innate immunity, signaling, stress responses, endospore attachment process and post-attachment behavioral modification of the juveniles. The expression profiles of fifteen selected transcripts were validated to be true by the qRT PCR. RNAi based silencing of transcripts coding for fructose bisphosphate aldolase and glucosyl transferase caused a reduction in endospore attachment as compared to the controls, whereas, silencing of aspartic protease and ubiquitin coding transcripts resulted in higher incidence of endospore attachment on the nematode cuticle.ConclusionsHere we provide evidence of an early transcriptional response by the nematode upon infection by Pasteuria prior to root invasion. We found that adhesion of Pasteuria endospores to the cuticle induced a down-regulated protein response in the nematode. In addition, we show that fructose bisphosphate aldolase, glucosyl transferase, aspartic protease and ubiquitin coding transcripts are involved in modulating the endospore attachment on the nematode cuticle. Our results add new and significant information to the existing knowledge on early molecular interaction between M. incognita and P. penetrans.

Project description: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.

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Transcriptomic changes in the pre-parasitic juveniles of Meloidogyne incognita induced by silencing of effectors Mi-msp-1 and Mi-msp-20.

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