4. Discussion
4.1 Identification of candidate genes which are specifically induced by chlorosis and wilting isolates
In this study an RNA-sequencing analysis ofV. dahliae challenged plants was performed that aimed at the identification of differentially expressed host genes involved in establishment of the chlorosis disease phenotype. In an RNA-sequencing experiment, a high number of replicates leads to more robust results (Auer and Doerge, 2010; Y., Liuet al., 2014; Schurchet al., 2016).
In this study, no biological replicates were included for Verticillium inoculated samples.
Instead, five chlorosis- and five wilting inducing isolates were treated as biological replicates in the differential gene expression analysis. It was assumed that all isolates of one interaction class trigger differential expression of a core set of symptom-specific host genes, which are causal to chlorosis or wilting symptom development. A differential expression analysis based on a biological replicate series of a single chlorosis or wilting reference isolate would also yield isolate-specific candidate genes that are differentially regulated in response to this particular isolate. Such candidates would for example comprise host genes regulated in response to lineage-specificVerticillium effectors acquired solely by the reference isolate. Kombrink et al.
(2017) recently described the Vd2LysM effector which is present in the V. dahliae isolate VdLs17 lineage-specific region but not in the genome of any other sequenced Verticillium isolate (Kombrink et al., 2017). The use of several isolates of one interaction class in the differential expression analysis allows subtraction of isolate-specific candidates, thus yielding only a core set of symptom-specific host genes required for the establishment of the chlorosis or wilting phenotype.
In order to analyse early host responses to Verticillium infection, i.e. during penetration and establishment of plant-pathogen interaction, the root transcriptome of two-week-oldA. thaliana Col-0 in vitro seedlings was analysed at 4 days post infection (dpi) by RNA-sequencing.
Previously conducted analyses of A. thaliana root transcriptome in response to the reference V. longisporum isolate c-VL43 show that substantial host transcriptional reprogramming takes place at this early time point (Ivenet al., 2012; J. Schmitz, PhD Thesis, 2015). Consistent with this extensive transcriptional reprogramming, 399 A. thaliana genes were significantly regulated (below a cut-off of FDR ≤ 0.05 and a log2 fold change in expression ≥ +1 and ≤ -1) during root infection by all ten V. dahliae isolates (data not shown). However, it has to be considered that these three transcriptome analyses are not strictly comparable. Infection
the infection assay performed in this thesis. In her transcriptome analysis, J. Schmitz (2015) analysed a different A. thaliana genotype compared to this thesis, in particular WTaos and WTcoi1-t, two wild-types back-crossed from the respective heterozygous mutant. Nevertheless, 18.1 % of genes differentially expressed in WTaos and 19.2 % in WTcoi1-t as well as 18.8 % of differentially expressed genes from the transcriptome analysis conducted by Iven et al.
(2012) overlap with candidates significantly regulated byV. dahliae isolates in this thesis (data not shown). Only few genes were differentially expressed specifically in response to chlorosis-or wilting-inducing isolates respectively at 4 dpi in A. thaliana roots (Table S1 and S2) indicating that transcriptional reprogramming induced by chlorosis or wilting isolates does not largely differ at this early stage of infection. This correlates to findings of Reuscheet al. (2014) which demonstrate that the V. longisporum reference chlorosis isolate c-VL43 and V. dahliae reference wilting isolate w-JR2 exhibit similar infection strategies and growth kinetics at early stages of infection. Both isolates enter the root at endodermis free zones and by 4 dpi colonize different cell layers including the central cylinder (Reuscheet al., 2014).
Late responses to Verticillium infection, i.e. during colonization of the xylem and the necrotrophic phase, were analysed in N. benthamiana shoot at 8, 12 and 16 dpi. In order to select candidates, differentially expressed genes were grouped into functional categories. Genes that either were most strongly regulated by chlorosis isolate infection within a functional category, i.e. G-type LecRLKAt5g24080 andRD17 or might represent putative key regulators in disease phenotype establishment, i.e. ANAC071 were chosen as candidates and further analysed in this study. However, further chlorosis isolate induced genes may play a role in establishment of the chlorosis disease phenotype. Among chlorosis-induced candidates, a number of genes encoding lipid or wax biosynthesis enzymes and lipid transfer proteins (LTPs) were present (Table S3, S4, S6 and S8). LTPs have been proposed to function in export of cuticular waxes, since LTP knock-down and knock-out lines demonstrate reduced amounts of certain wax components in theArabidopsis cuticula (DeBonoet al., 2009; Leeet al., 2009; Kim et al., 2012). Interestingly, tree tobacco (Nicotiana glauca) was shown to increase wax deposition under drought stress and leaves from drought stressed plant were more resistant to water loss (Cameron et al., 2006). Moreover, Arabidopsis LTP3 was demonstrated to be implicated in drought responses, since ltp3 loss of function mutants showed reduced drought
tolerance (Guo et al., 2006; S., Zhou et al., 2012; Xu et al., 2014; Zhuang et al., 2015). Five aquaporin genes belonging to the plasma membrane- and tonoplast-intrinsic protein families were up-regulated during chlorosis isolate infection at 8, 12 and 16 dpi as well as in the collective analysis of 8, 12 and 16 dpi (Table S3, S4, S6 and S8). Five aquaporin genes belonging to these families were also induced by the V. longisporum chlorosis isolate c-VL43 infection ofA. thaliana at 18 dpi in a microarray experiment conducted by H. Tappe. PIP2;2 (PLASMA MEMBRANE INTRINSIC PROTEIN 2;2) and TIP2;2 (TONOPLAST INTRINSIC PROTEIN 2;2) genes were up-regulated in both transcriptome analyses (H.Tappe, PhD thesis, 2008). None of the differentially expressed aquaporin genes was selected as candidate for further analysis, since members of Arabidopsis aquaporin subfamilies are known to be highly homologous. Members of the plasma membrane intrinsic protein subfamily share 71.8 to 97.2 % sequence homology at the amino acid level, whereas tonoplast intrinsic protein subfamily members share 44.1 to 93.1 % amino acid sequence homology (Quigleyet al., 2002).
Thus, it is likely that aquaporins function redundantly, which makes reverse genetic analysis difficult. Furthermore, several drought responsive genes including RD17 and ERF53 were up-regulated by chlorosis isolate infection (Table S3, S6 and S8). Together these results suggest that drought tolerance during chlorosis isolate infection rather results from genes belonging to distinct families than from a single gene or members of one gene family.
Reusche et al. (2014) observed a stronger induction of the drought marker gene RD29B (Yamaguchi-Shinozaki and Shinozaki, 1993) in A. thaliana plants infected with the wilting isolate w-JR2 as compared to the chlorosis isolate c-VL43 during concomitant drought stress.
Yet, RD29B was not expressed in the watered control A. thaliana plants during Verticillium challenge (Reuscheet al., 2014). Interestingly,RD29B as well as the drought and osmotic stress signalling components SNRK2.9 (SNF1-RELATED PROTEIN KINASE 2.9) and SNRK3.14 (SNF1-RELATED PROTEIN KINASE 3.14) (Tripathiet al., 2009; Fujiiet al., 2011; Tsouet al., 2012; Chen et al., 2013) were up-regulated in normally watered N. benthamiana by wilting isolate infection (Table S2 and S9). This result implies that proliferation of wilting isolates is sufficient to trigger drought stress in N. benthamiana without water withdrawal. As described in section 1.4, water stress may potentially result from clogging of water conducting xylem vessels due to pathogen proliferation, formation of vascular gels and tyloses.