Understanding the molecular response of potato during infections with Pectobacterium

Abstract

Pectobacterium atrosepticum (Pba SCRI1043) and Pectobacterium carotovorum subsp. brasiliensis (Pbr ICMP19477) are considered necrotrophic/hemibiotrophic bacterial plant pathogens belonging to the Enterobacteriaceae family. They are responsible for blackleg disease on potato stems during the growing season and soft rot of tubers post-harvest. Coronafacic acid (CFA) is a virulence determinant in these Pectobacterium taxa, supporting the development of blackleg in potato stems, but little is known about its role during infection of potato tubers or the mechanisms associated with its activity. As Pba and Pbr are seed borne pathogens whose spread is primarily dependent on contaminated tubers, determining the molecular interactions that occur between pathogen and host in potato tubers and understanding the role of important virulence factors such as CFA in this process, are vital to implementing effective control strategies. In this study, RNA sequencing technology was used to examine the transcriptional response in susceptible potato tubers (‘Summer Delight’) challenged with Pba SCRI1043 or Pbr ICMP19477. In addition, to begin to understand the function of CFA, the transcriptional response of the host to Pba SCRI1043 was compared to that elicited upon infection with a mutant unable to produce CFA.

In the first part of the study, the most appropriate software available for detection of differential expression in potato was established by comparing the quality of the differential expression data produced by two software packages, DESeq2 and Cuffdiff2. Comparison of the read counts and fold change data from both software packages confirmed the detection of fewer false positives using DESeq2. Based on this finding, DESeq2 was chosen as the most robust approach for differential expression detection. Subsequent comparison of the potato tuber transcriptome following infection by either Pbr ICMP19477 or Pba SCRI1043 revealed that the number of differentially expressed genes was consistently higher across 6, 12 and 24 hours post inoculation in response to Pbr ICMP19477. Furthermore, the magnitude of the transcriptional change (as determined by log2 fold change values) observed in potato tubers was consistently higher upon inoculation with Pbr ICMP19477 than in response to Pba SCRI1043. Consistent with the production of plant cell wall degrading enzymes by both pathogens, infection with Pbr ICMP19477 and Pba SCRI1043 induced a characteristic damage associated molecular patterns-mediated pattern-triggered immunity (PTI) defence response. In particular, genes involved in the early PTI defence response including the PI-PLC signalling pathway, oxidative burst and ethylene (ET) biosynthesis and signalling were significantly differentially expressed. Secondary metabolism was also induced; genes involved in the synthesis of terpenoids showing significant up regulation in response to both soft rot Enterobacteriaceae (SRE). SRE infection also led to up regulation of genes related to phytoalexins biosynthesis, including divinyl ether synthase, which is involved in the synthesis of the antimicrobials colneleic and colnelenic acid. Interestingly, no differential expression of genes relating to jasmonic acid (JA) biosynthesis was observed, although the genes encoding MYC2 and JAZ, related to downstream JA signalling, were significantly induced indicative of JA-independent MYC2 activation. Pbr ICMP19477 appeared to elicit additional specific responses by the host, with the two snakin genes Sn-1 and Sn-2 amongst those genes with greatest up regulation in response to this pathogen. Sn-1 and Sn-2 encode well known antimicrobial peptides with activity against SRE. A specific cluster of LRR genes also seemed to be up regulated in response to pectobacteria, indicative of the host initiating an effector-triggered immunity during the susceptible interaction with this pathogen.

In Pba SCRI1043 and Pbr ICMP19477, the biosynthetic gene clusters responsible for CFA biosynthesis are harboured on closely related horizontally acquired islands (HAIs). Interestingly, loss of the HAI (HAI2) in Pba SCRI1043 resulted in very few observable changes in differential expression in potato tubers, even though the CFA cluster it harbours enhances infection of the stem. The notable exception to this general lack of differential expression when compared to wild type was the expression of MYC2. Specifically, no differential expression of MYC2 was observed upon infection with the CFA mutant when compared to the non-inoculated control whereas MYC2 was induced in response to Pba SCR1043 (and Pbr ICMP19477). This result suggested that CFA might target the MYC2-mediated JA response, which alters the production of key defence-related products required to combat bacterial infection. This is consistent with the targeting of the MYC2-JAZ complex by coronatine, a related toxin produced by taxa of Pseudomonas syringae.

To study how CFA might influence virulence further, potato tubers were also exposed to exogenous CFA and their transcriptional response was subsequently compared to those of the tubers treated with SRE or the non-inoculated control. These comparisons showed a significant overlap in differential expression upon exposure to CFA and the SRE, with CFA impacting 40% of the genes that responded to inoculation with the bacteria. In particular, application of exogenous CFA resulted in differential expression of the JA and ET signalling pathways in much the same way as inoculation with Pba SCRI1043 and Pbr ICMP19477. In addition, JAR1 was up regulated in response to exogenous CFA at 6 hpi. JAR1 encodes JA-amino acid synthase, an enzyme involved in the conjugation of JA to amino acids. Given that CFA is a molecular mimic of jasmonate, this result suggested that the presence of CFA induces its conjugation to an unknown amino acid. It is probable that the conjugate is necessary for downstream activation of JA signalling and, in particular, differential expression of MYC2. Consistent with this, mass spectroscopy of extracts from tubers and stems infected with either Pba SCRI1043 and Pbr ICMP19477 confirmed the production of both CFA and its amino acid conjugate CFA-valine during infection.

In summary, this thesis presents new information on the defence response deployed by potato to two important SRE pathogens and novel insights into CFA-mediated host susceptible.