Gene expression responses of tomato inoculated with Pectobacterium carotovorum subsp. carotovorum

Abstract Defense responses of tomato (Solanum lycopersicum L.) against attack by Pectobacterium carotovorum subsp. carotovorum (Pcc), the causal agent of soft rot diseases, were studied. The expression of some tomato defense genes were evaluated by real‐time PCR quantification analysis, 24 and 72 hr after actively growing tomato plants were inoculated with Pcc. These included: MYB transcriptor factor, ethylene response element‐binding protein, suppressor of the G2 allele of Skp1, cytochrome P450, small Sar1 GTPase, hydroxycinnamoyl‐CoA:quinate hydroxycinnamoyl transferase, pathogenesis‐related protein 1a, endo‐1,3‐beta‐glucanase, chitinase, proteinase inhibitor, defensin, CC‐NBS‐LRR resistance protein, and phenylalanine ammonia lyase. The results showed dynamic transcriptomic changes, with transcripts exhibiting different expression kinetics at 24 and 72 hr to confer resistance to tomato against Pcc infection.


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tomato leaf. In total, 15 plants were included in the study. Four whole leaves were infiltrated per plant.
The study consisted of five treatments. Four sets of three plants were either inoculated with buffer or with the bacterium and harvested after 24 and 72 hr. A final negative control was also included, with leaves from three untreated plants harvested at the beginning of the experiment. Once harvested, all leaves were kept at −80°C and then the frozen samples were ground in liquid nitrogen using a mortar and pestle.
Total RNA was extracted from 100 mg ground leaf tissue using the TRIzol® Reagent method (Invitrogen). The extracted RNA was treated with DNase I (Thermo Scientific). From the total RNA, cDNA was synthesized using a RevertAid™ Premium First Strand cDNA synthesis kit (Fermentas, Thermo Scientific). For accurate calculation of relative gene expression by qPCR, the input RNA was stan- Information on the primers used appears on Appendix Table A1.
The experiment was performed using three biological replicates (repeats) in plants. One biological replicate was a pool of four inoculated leaves from different plants of a single genotype. This approach was recommended by Brady et al. (2015) because independent replication is the foundation of any successful hypothesis test; instead of repeatedly sampling the same individual, it is better to sample multiple individuals to minimize the potential for bias in the analysis. The melting curve of the qPCR, always showed a single peak confirming that a single amplicon had been generated by qPCR with no other unspecific amplification which could be from a contaminating genomic DNA. Quantification of the relative changes in gene expression was performed using the relative standard curve method (Djami-Tchatchou et al., 2015;Djami-Tchatchou, Ncube, Steenkamp, & Dubery, 2017) with elongation factor 1-alpha and actin 8 used as references genes. Datasets were statistically compared between non-treated samples and treated samples at each time point using one-way analysis of variation with the statistical analysis software GraphPad inStat 3 (GraphPad Software). The confidence level of all analyses was set at 95%, and values with p < .05 were considered significant.

| RE SULTS AND D ISCUSS I ON
The extracted RNA was found to be pure and undegraded, and furthermore, changes to the transcriptome were dynamic, with transcripts exhibiting different expression kinetics at 24 and 72 hr following the inoculation of tomato with P. carotovorum subsp. carotovorum (Pcc) ( Table 1). The fold expression varied from relatively low (>2 fold) to high (>10 fold) compared with the basal levels of nontreated cells (Figure 1a-m).
The gene expression analysis showed that at 24 hr following bacterial inoculation, the expression profiles of some genes, namely,  tions. This study was not an exhaustive investigation of the genetic defense mechanisms of tomato against Pcc but a preliminary investigation to assess overall response to create a platform for future studies.
During plant-pathogen interactions, EREBP has been shown to be intimately related to defense responses, stress signaling pathways (Wang, Li, & Ecker, 2002); and the level of the biosynthesis of ethylene increases rapidly leading to the transcription of some defenserelated genes such as β-1,3-glucanase and chitinase class I (Sharma et al., 2010).    (PR12) and (m) Cc-nbs-lrr resistance protein. The data was normalized using Elf α and 18S to give the relative gene expression. Each data point is the average of 3 biological replicates, and error bars represent the SEM between biological replicates. Results were analyzed using ANOVA, with confidence level of 95%, followed by a Tukey's post-test. Same letter indicates no significant difference and different letters indicate significant difference between samples with p < .05 SGT1 regulates defense responses triggered by various pathogens, and it has been found from previous findings that SGT1 is involved in plant resistance to pathogen attack (Djami-Tchatchou et al., 2015;Meldau, Baldwin, & Wu, 2011). In this study, we found that the transcripts of SGT1 exhibited an upregulation at 72 hr postinoculation. SGT1 was first found to confer resistance to Peronospora parasitica in Arabidopsis , and the SGT1 gene interacts with RAR1 (required for Mla12 resistance), to confer resistance by multiple R genes recognizing distinct avirulent P. parasitica or Pseudomonas syringae pv. tomato isolates .
Plant CytP450 has been shown to be involved in various biochemical pathways to produce primary and secondary metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates as well as plant hormones (Mizutani, 2012). SAR1-GTPase is a small monomeric GTP-binding protein be- in CGA accumulation (Lepelley et al., 2007;Rommens et al., 2008).
Our results indicate that HQT is involved in the response of tomato to Pcc infection with a positive regulation with MYB transcription factor. HQT is also known to be correlated with phenylalanine ammonia-lyase (PAL), the first enzyme in the phenylpropanoid pathway linking primary metabolism to secondary metabolism (Tohge, Watanabe, Hoefgen, & Fernie, 2013). We found that the transcript of PAL was not differentially expressed at 24 hr with a slight non- PR6 was produced to restrict P. syringae spread in tomato (Koiwa, Bressan, & Hasigawa, 1997). Our results indicate that the upregulation of PR12 at 72 hr was to enhance tomato resistance to Pcc.
The noninduction of PR1 observed in this study showed that it is not involved in the response of tomato to Pcc. In this study, genes of interest were selected based on the fact that they were previously reported to be involved in tomato (or related family) defense response to pathogen attack (Alfano et al., 2007;Block et al., 2005;Djami-Tchatchou et al., 2015;Hafez et al., 2013;Medeiros et al., 2009). We conclude that Pcc infection of the tomato triggers the expression of a number of the genes selected, which is an indication of their involvement in defense. However, this preliminary finding requires further investigation such as the use of knockout tomato mutants to comprehensively assess gene function and the defense response.

ACK N OWLED G M ENTS
This work was performed with funding provided by the University

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
ATD was involved in plant inoculation, sample collection, and performed the real-time PCR and analyzed and interpreted gene expression data. LBT was involved in concept formulation and project administration. KN was involved in concept formulation, preparation of bacterial inoculum, plant inoculation, and sample collection. ATD, LBT, and KN were all involved in manuscript preparation each writing a section. Review and editing was done by all.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article.