Search

Supplementary Material 1

Root-knot nematode (RKN, Meloidogyne javanica) presents a great challenge to Solanaceae crops, including the potato. In this report, we conducted an investigation to understand the transcriptional regulation of molecular responses in potato roots during a compatible interaction following RKN infection. In this study, analysis of gene expression profiles using RNA-seq of Solanum tuberosum cv Mondial with RKN interaction at 0, 3- and 7-days post-inoculation (dpi). In total, 4,948 and 4,484 genes were respectively detected as differentially expressed genes (DEGs) at 3 and 7 dpi. Functional annotation revealed that genes associated with metabolic process were enriched at the transcriptional level suggesting they have an important role in RKN disease development. Nematode infection caused down-regulation of 282 genes associated with pathogen perception hence interfering with activation plant immune system. Further, late activation of pathogenesis-related genes, down-regulation disease resistance genes and activation of host antioxidant system contributed to a susceptible response. Activation of Jasmonic acid (JA) pathway and protease inhibitors was due to wounding during nematode migration and feeding. Nematode infection suppressed ethylene (ET) and salicylic acid (SA) signalling pathway hindering SA/ET responsive genes involved with defense. Induction of auxin biosynthesis genes, regulation of cytokinin levels and up-regulation of transporter genes facilitated of nematode feeding sites (NFSs) initiation. The regulation of several families of transcription factors (TFs) in the plant, such as WRKY, GRAS, ERF BHLH and MYB, was affected by RKN infection disrupting plant defense signalling pathways. This clearly suggest that TFs played an indispensable role in physiological adaptation for successful RKN disease development. This genome-wide analysis revealed the molecular regulatory networks in potato roots which are successfully manipulated by RKN. Being the first study analysing transcriptome profiling of RKN diseased potato, it will provide unparalleled insight into the mechanism underlying disease development.

In this study, we examine the impact of transcriptional network rearrangements driven by horizontal gene acquisition in PhoP and SlyA regulons using as a case study the phytopathosystem comprised of potato tubers and the soft rot pathogenPectobacterium carotovorumsubsp.brasiliense(Pcb1692). By comparing those two networks with that of PecS obtained from the closely relatedDickeya dadantii, we found that: (a) 24-31% of the genes regulated at late infection are genus-specific (GS) (based on Pectobacterium and Dickeya genera), and that (b) of these, 28.1-44.4% were predicted with high confidence as horizontal gene transfer (HGT) candidates. Further, genome simulation and statistical analyses corroborated the bias in late infection regulons towards the transcriptional control of candidate GS-HGT genes by PhoP, SlyA, and PecS, highlighting the prominence of network rearrangements in these late infection regulons. The evidence further supports the circumscription of two horizontally acquired quorum sensing regulators (carR andexpR1) by the PhoP network. By recruitingcarR andexpR1, the PhoP network also impacts certain host adaptation- and bacterial competition-related systems, seemingly in a quorum sensing-dependent manner, such as the type VI secretion system, carbapenem biosynthesis, and plant cell walls degrading enzymes (PCWDE) such as cellulases and pectate lyases. Conversely, polygalacturonases and the type III secretion system (T3SS) exhibit a transcriptional pattern that suggests quorum sensing-independent regulation by the PhoP network. This includes a yet uncharacterized novel phage-related gene family within the T3SS gene cluster that has been recently acquired by two Pectobacterium species. The evidence further suggests a PhoP-dependent regulation of carbapenem and PCWDE-encoding genes based on the synthesized products’ optimum pH. The PhoP network also controlsslyA expressionin planta, which seems to impact the carbohydrate metabolism regulation, especially at early infection when 69.6% of the SlyA-regulated genes from that category also require PhoP to achieve normal expression levels.AUTHOR SUMMARYExchanging genetic material through horizontal transfer is a critical mechanism that drives bacteria to efficiently adapt to host defenses. In this report, we demonstrate that a specific plant pathogenic species (from the Pectobacterium genus) successfully integrated a population density-based behaviour system (quorum sensing) acquired through horizontal transfer into a resident stress-response gene regulatory network controlled by the PhoP protein. Evidence found here underscores that subsets of bacterial weaponry critical for colonization, typically known to respond to quorum sensing, are also controlled by PhoP. Some of these traits include different types of enzymes that can efficiently break plant cell walls depending on the environmental acidity level. Thus, we hypothesize that PhoP ability to elicit regulatory responses based on acidity and nutrient availability fluctuations may have strongly impacted the fixation of its regulatory connection with quorum sensing. In addition, another global gene regulator known as SlyA was found under the PhoP regulatory network. The SlyA regulator controls a series of carbohydrate metabolism-related traits, which also seem to be regulated by PhoP. By centralizing quorum sensing andslyA under PhoP scrutiny, Pectobacterium cells added an advantageous layer of control over those two networks that potentially enhances colonization efficiency.

List of primers used for RT-qPCR validation of RNA-seq expression data (DOCX 12Â kb)

RT-qPCR confirmation of five potato defense-related miRNAs in S. tuberosum cv BP1, computationally predicted to target some of the lincRNA transcripts. U6 snRNA was used as the reference gene. The fold changes of miRNAs at each time point were calculated relative to calibrator (control sample; 0 hpi). The experiments were done in triplicate. Error bars represent the fold change range calculated by 2-(ΔΔCt±SD). (PDF 86 kb)

List of primers used for RT-PCR validation of novel sRNAs (DOCX 12Â kb)

List of primers used for RT-qPCR validation of RNA-seq expression data (DOCX 12Â kb)

List of primers used for RT-PCR validation of novel sRNAs (DOCX 12Â kb)

RT-qPCR confirmation of five potato defense-related miRNAs in S. tuberosum cv BP1, computationally predicted to target some of the lincRNA transcripts. U6 snRNA was used as the reference gene. The fold changes of miRNAs at each time point were calculated relative to calibrator (control sample; 0 hpi). The experiments were done in triplicate. Error bars represent the fold change range calculated by 2-(ΔΔCt±SD). (PDF 86 kb)

A) Comparison of the genomic distribution of lincRNAs and protein-coding genes across the 12 potato chromosomes. The outer grey track represents the 12 potato chromosomes, with a scale (Mb) showing the length of each chromosome. The red histograms (second track with an outer orientation) and blue histograms (third track with inner orientation) represent the abundance and distribution of mRNA and lincRNAs, respectively, throughout the potato genome. The bin size (histogram width)â =â 5 Mbp). B) Comparison of LincRNA lengths to protein-coding mRNA transcripts in potato (PGSC_DM_v4.03 genome assembly). (PDF 258Â kb)

A) Comparison of the genomic distribution of lincRNAs and protein-coding genes across the 12 potato chromosomes. The outer grey track represents the 12 potato chromosomes, with a scale (Mb) showing the length of each chromosome. The red histograms (second track with an outer orientation) and blue histograms (third track with inner orientation) represent the abundance and distribution of mRNA and lincRNAs, respectively, throughout the potato genome. The bin size (histogram width)â =â 5 Mbp). B) Comparison of LincRNA lengths to protein-coding mRNA transcripts in potato (PGSC_DM_v4.03 genome assembly). (PDF 258Â kb)

Comparison of the 1113 lincRNA transcripts identified in the present study with potato lncRNAs available in the GreenC database. (PDF 105Â kb)