3. Results
3.1 Host plant genes which are differentially regulated by Verticillium chlorosis or wilting isolate infection
Verticillium longisporum induces substantial developmental reprogramming of its host plant Arabidopsis thaliana leading to transdifferentiation of chloroplast-containing bundle sheath cells to functional xylem elements. Furthermore, re-initiation of cambial activity and transdifferentiation of xylem parenchyma cells result in xylem hyperplasia within the Arabidopsis vascular system. Thede novo xylem formation is associated with enhanced water storage capacity and enhanced drought tolerance ofV. longisporuminfected plants (Reuscheet al., 2012).
Induction ofde novo xylem formation is not restricted toV. longisporum. In a recent study, the interaction phenotypes of A. thaliana Col-0 with 47 V. dahliae isolates were systematically analysed. Among compatible interactions fiveV. dahliae isolates were described which trigger V. longisporum-like symptoms including de novo xylem formation, stunted growth, leaf chlorosis and early senescence. In marked contrast, 36 isolates caused wilting, stunted growth and decay of older rosette leaves (K. Thole, PhD thesis, 2016). In the following, these clearly distinguishable disease phenotypes will be referred to as “chlorosis” and “wilting”.
Chlorosis-inducingVerticillium isolates will be marked with the prefix “c” and wilting isolates with the prefix “w”. It was postulated that these disease phenotypes are triggered by lineage-specific Verticillium effector molecules, which induce distinct transcriptional and developmental reprogramming patterns of the host plant. Several V. dahliae chlorosis isolate specific, putatively secreted candidate effectors are transcriptionally inducedin plantaand may possibly trigger the chlorosis disease phenotype (K. Thole, PhD thesis, 2016).
This study aimed at the identification of differentially expressed plant host genes that in response to putative effectors may be involved in establishment of the chlorosis disease phenotype. Therefore, same RNA-sequencing data as employed by K. Thole for the identification of chlorosis isolate specific fungal effectors was used. In her RNA-Sequencing experiment, K. Thole spray inoculated two-week-old A. thaliana Col-0in vitro seedlings with
responses to Verticillium infection, i.e. during colonization of the xylem and the necrotrophic phase, were assessed. N. benthamiana was chosen as host plant for the analysis of late time points of infection, since it accumulates higher amounts of Verticillium biomass compared to A. thaliana (Faino et al., 2012). Sufficient amounts of fungal biomass were essential, in order to obtain high quality RNA-sequencing data of the fungal transcriptome. As in A. thaliana Col-0, chlorosis-inducingVerticillium isolates trigger bundle sheath cell transdifferentiation in N. benthamiana, whereas wilting isolates do not (K. Thole, PhD thesis, 2016), suggesting that chlorosis isolate infection leads to similar transcriptional and developmental reprogramming of this solanaceous host. Plants were infected with five chlorosis-inducing as well as five wilting-inducingV. dahliae isolates (Table 1), genome sequence of which was either published in previous studies orde novo assembled by K. Thole (Klostermanet al., 2011; de Jongeet al., 2012; de Jonge et al., 2013; K. Thole, PhD Thesis, 2016). For each time point, three mock treated controls were analysed.
Table 1. Verticillium isolates used in A. thaliana and N. benthamiana infection for RNA-sequencing analysis.
Verticillium species Isolate Symptoms on A. thaliana Col-0 Symptom intensity*
V. dahliae c-V76 chlorosis ++
V. dahliae c-V138I chlorosis ++
V. dahliae c-T9 chlorosis ++
V. dahliae c-V781I chlorosis +++
V. dahliae c-ST100 chlorosis +++
V. dahliae w-V192I wilting -/+
V. dahliae w-VdLs17 wilting +
V. dahliae w-JR2 wilting +++
V. dahliae w-DVD-S29 wilting +++
V. dahliae w-DVD-31 wilting +++
* macroscopically determined intensity of disease symptom development as described in K. Thole, PhD thesis, 2016: mild symptoms (-/+), moderate symptoms (+), strong symptoms (++), very strong symptoms (+++).
Preparation of RNA-samples, quality control, RNA-sequencing and RNA-read mapping were performed by the Transcriptome and Genome Analysis Laboratory (TAL, Department of Developmental Biochemistry, Georg August University Göttingen, Göttingen, Germany) using the Illumina HiSeq 2000 system. RNA-sequencing reads obtained from the A. thaliana 4 dpi root samples were mapped to the A. thaliana TAIR10 genome release (Berardiniet al., 2015), whereas reads obtained from theN. benthamiana 8, 12 and 16 dpi shoot samples were mapped
RNA-read counts were received from TAL and subjected to differential gene expression analysis usingRobiNA v1.2.4 (Lohseet al., 2012) and the DESeq analysis method (Anders and Huber, 2010).
Differential gene expression analysis performed in my PhD thesis aimed at the identification of plant genes, which are specifically up- or down-regulated during infection with the chlorosis-or wilting-inducing V. dahliae isolates, since these genes may be implicated in establishment of the respective disease phenotype. In order to find host genes, which are significantly regulated during infection with chlorosis-inducing isolates, raw RNA-read counts of the chlorosis group samples were compared to the read counts of the wilting and mock group.
Genes which were up- or down-regulated with a False Discovery Rate (FDR) of ≤ 0.05 and a log2 fold (L2F) change in expression of ≥ +1 and ≤ -1 in the chlorosis group but not the wilting and mock group were considered as significantly regulated. Host genes, which are significantly regulated during infection with wilting-inducing isolates were identified with the same approach. Here, the wilting group was compared to the chlorosis and mock group. A complete list of significantly regulated genes at 4 days post infection of A. thaliana roots and 8, 12 and 16 days post infection of N. benthamiana shoots with V. dahliae chlorosis-inducing or wilting-inducing isolates is shown in Supplementary Tables S1-S7.
Disease symptom intensities and infection kinetics often vary between single infection experiments. It is conceivable that a gene appears to be regulated at different time points due to variabilities in kinetics between infection experiments. In order to avoid loss of differentially regulated genes due to variabilities in infection kinetics, raw RNA-read counts of 8, 12 and 16 dpiN. benthamiana shoot samples were added and collectively subjected to differential gene expression analysis as described above. A complete list of significantly regulated genes during infection with chlorosis- or wilting-inducing V. dahliae isolates generated in the collective analysis of 8, 12 and 16 dpiN. benthamiana shoot samples is shown in Supplementary Tables S8 and S9.
In order to select candidates for further analyses, differentially expressed genes were grouped into functional categories based on MapMan (Thimmet al., 2004) BIN names and published literature if available (Table S1-S9, right column). A. thaliana homologs were assigned to N. benthamiana genes using the N. benthamiana transcripts annotation file (version 0.4.4) and
modification, transdifferentiation, signalling, water transport and drought response were of special interest, since they may play a potential role in the induction of the chlorosis phenotype characterised byde novo xylem formation and enhanced drought tolerance.
Table 2. List of selected candidate genes differentially regulated during infection withchlorosis-inducing Verticillium isolates.Differential gene expression was analysed usingRobiNA v1.2.4 (Lohseet al., 2012) and the DESeq analysis method (Anders and Huber, 2010) between samples of thechlorosis group versuswilting group and mock treatment. Raw P-values were adjusted using the False Discovery Rate algorithm (Benjamini and Hochberg, 1995). Positive L2F change in expression, representing gene induction, is highlighted in shades of red, whereas negative L2F change in expression, representing gene repression, is highlighted in shades of blue. Gene function is colour-coded.
(Nb) Description of At homolog Functions in
NbS00002660g0010 AT5G24080 1.6 4.0 4.8 5.4
Protein Kinase Family Protein signalling
NbS00034147g0011 AT4G17980 0.2 2.7 3.8 2.3 ANAC071 (Arabidopsis NAC domain containing protein 71)
transdifferentiation (tissue reunion)
NbC25873455g0003 AT1G20440 -0.3 5.5 5.6 5.5 RD17; COR47
(COLD-REGULATED 47) drought response
Three candidate genes, which are differentially regulated during infection with chlorosis-inducing Verticillium isolates, were chosen for further analyses (Table 2). Firstly, NbS00002660g0010, aN. benthamiana gene which is homologous toA. thaliana protein kinase geneAt5g24080was chosen.NbS00002660g0010 was selected, because it was the only protein kinase gene significantly regulated and highly induced by chlorosis isolate infection at 8 and 12 dpi as well as in the collective analysis of 8, 12 and 16 dpi (Table S3, S4 and S8). Moreover, its A. thaliana homolog At5g24080 was 1.6 log2 fold, yet not significantly, induced in A. thaliana chlorosis isolate root infection at 4 dpi (Table 2). No molecular analyses of At5g24080 were published so far (November 2017). As a protein kinase, it may be implicated in signal transduction required for the establishment of the chlorosis phenotype. Secondly, NbS00034147g0011, a N. benthamiana gene which is homologous to the A. thaliana transcriptional factor ANAC071 (Arabidopsis NAC domain containing protein 71).
NbS00034147g0011 was selected, since expression of another NAC domain transcription factor,VND7 (Vascular Related NAC-Domain Protein 7) is known to be required forde novo xylem formation during infection with the chlorosis-inducing V. longisporum isolate c-VL43 (Reusche et al., 2012). Additionally, ANAC071 was shown to be required for vascular tissue proliferation during graft reunion in hypocotyl of Arabidopsis seedlings (Matsuoka et al., 2016). As a consequence, it is conceivable that ANAC071 may be involved in vascular tissue
NbC25873455g0003, a N. benthamiana gene which is homologous to A. thaliana RD17 (Responsive to Desiccation 17) was chosen. NbC25873455g0003 was selected, because it was the only drought responsive gene significantly regulated and highly induced by chlorosis isolate infection at 8 and 16 dpi as well as in the collective analysis of 8, 12 and 16 dpi (Table S3, S6 and S8). Its A. thaliana homolog RD17 belongs to the dehydrin protein family, which is generally assumed to play a pivotal role in protection of the plant cell during dehydration (Hanin et al., 2011). For this reason, this gene may be involved in enhanced drought tolerance during chlorosis isolate infection.