A transcriptomic snapshot of early molecular communication between Pasteuria penetrans and Meloidogyne incognita

© The Author(s). 2018Background: Southern 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. Results: A 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. Conclusions: Here 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.Peer reviewedFinal Published versio

Dynamics of Membrane Potential Variation and Gene Expression Induced by <em>Spodoptera littoralis</em>, <em>Myzus persicae</em>, and <em>Pseudomonas syringae</em> in Arabidopsis

<div><h3>Background</h3><p>Biotic stress induced by various herbivores and pathogens invokes plant responses involving different defense mechanisms. However, we do not know whether different biotic stresses share a common response or which signaling pathways are involved in responses to different biotic stresses. We investigated the common and specific responses of <em>Arabidopsis thaliana</em> to three biotic stress agents: <em>Spodoptera littoralis</em>, <em>Myzus persicae</em>, and the pathogen <em>Pseudomonas syringae</em>.</p> <h3>Methodology/Principal Findings</h3><p>We used electrophysiology to determine the plasma membrane potential (V_m) and we performed a gene microarray transcriptome analysis on Arabidopsis upon either herbivory or bacterial infection. V_m depolarization was induced by insect attack; however, the response was much more rapid to <em>S. littoralis</em> (30 min −2 h) than to <em>M. persicae</em> (4–6 h). <em>M. persicae</em> differentially regulated almost 10-fold more genes than by <em>S. littoralis</em> with an opposite regulation. <em>M. persicae</em> modulated genes involved in flavonoid, fatty acid, hormone, drug transport and chitin metabolism. <em>S. littoralis</em> regulated responses to heat, transcription and ion transport. The latest Vm depolarization (16 h) was found for <em>P. syringae</em>. The pathogen regulated responses to salicylate, jasmonate and to microorganisms. Despite this late response, the number of genes differentially regulated by <em>P. syringae</em> was closer to those regulated by <em>S. littoralis</em> than by <em>M. persicae</em>.</p> <h3>Conclusions/Significance</h3><p>Arabidopsis plasma membranes respond with a V_m depolarization at times depending on the nature of biotic attack which allow setting a time point for comparative genome-wide analysis. A clear relationship between V_m depolarization and gene expression was found. At V_m depolarization timing, <em>M. persicae</em> regulates a wider array of Arabidopsis genes with a clear and distinct regulation than <em>S. littoralis</em>. An almost completely opposite regulation was observed between the aphid and the pathogen, with the former suppressing and the latter activating Arabidopsis defense responses.</p> </div

GO analysis of Arabidopsis genes regulated by <i>Myzus persicae</i> herbivory.

<p>Red color indicates up-regulation, green color indicates down-regulation.</p

Plasma transmembrane potential (V_m) depolarization measured in Arabidopsis mesophyll leaves at different times upon herbivory by <i>Spodoptera littoralis</i> and <i>Myzus persicae</i> and infection by <i>Pseudomonas syringae</i>.

<p>Chewing herbivore induces a fast V_m depolarization that lasts about 4–6 h from feeding, whereas phloem feeding induces a V_m depolarization that occurs after about 6 h from feeding. Infection by <i>P. syringae</i> causes a V_m depolarization about 16 h after infection. No matter the biotic stress the level of the highest V_m depolarization shows the same value (statistical significance P>0.05). For each time point at least 50 measurements were performed. The timing of V_m depolarization depends on biotic damage. Bars represent standard error, different letters indicate significant (P<0.05) differences.</p

Cluster analysis of commonly regulated genes in Arabidopsis fed by the herbivores <i>Myzus persicae</i> and <i>Spodoptera littoralis</i> and infected by <i>Pseudomonas syringae</i>.

<p><i>M. persicae</i> shows a lower statistical linkage with <i>S. littoralis</i> and <i>P. syringae</i>, which are linked together.</p

GO analysis of Arabidopsis genes regulated by <i>Pseudomonas syringae</i> infection.

<p>Red color indicates up-regulation, green color indicates down-regulation.</p

GO analysis of Arabidopsis commonly expressed genes upon <i>Pseudomonas syringae</i> (<i>PS</i>) infection and <i>Spodoptera littoralis</i> (<i>SL</i>) herbivory.

<p>Red color indicates up-regulation, green color indicates down-regulation.</p

<i>Arabidopsis thaliana</i> genes commonly expressed at the time of Vm depolarization upon <i>Spodoptera littoralis</i> (2 h) and <i>Myzus persicae</i> (5 h) herbivory and <i>Pseudomonas syringae</i> (16 h) infection.

<p>Values are expressed as fold change with respect to controls (P<0.05). AGI, Arabidopsis Genome Initiative gene index.</p

<i>Arabidopsis thaliana</i> genes differentially expressed at the time of Vm depolarization (2 h) upon <i>Spodoptera littoralis</i> herbivory.

<p>Values are expressed as fold change with respect to controls (P<0.05). AGI, Arabidopsis Genome Initiative gene index.</p

Venn diagram of commonly and differentially expressed Arabidopsis genes upon herbivory by <i>Spodoptera littoralis</i> and <i>Myzus persicae</i> and infection by <i>Pseudomonas syringae</i>.

<p>Venn diagram of commonly and differentially expressed Arabidopsis genes upon herbivory by <i>Spodoptera littoralis</i> and <i>Myzus persicae</i> and infection by <i>Pseudomonas syringae</i>.</p