Transcriptional analysis of chitosan-induced resistance
Priming of gene expression normally follows a characteristic pattern:
differential expression is low, transient or often non-detectable after
treatment with the elicitor only (i.e. Chitosan + Mock) and enhanced
differential expression occurs upon subsequent infection (i.e. Chitosan
+ B. cinerea ) compared to infected plants that were not
pretreated with the chemical (i.e. Water + B. cinerea ) (Conrath
et al., 2006, Martinez-Medina et al., 2016). Importantly, the expression
kinetics are also key points for the establishment of priming. To
further determine the priming basis of chitosan-induced resistance, we
performed whole transcriptomic analysis at 6, 9 and 12 hours post
infection (hpi) with B. cinerea. These time points were selected
as they cover the early, non-symptomatic start of the B. cinereainfection process. Unsupervised data analysis was first performed to
observe global changes in the experiment. For this, we did a 2D
principal component analysis (PCA) at different hours post infection.
This analysis shows that chitosan treatments did not trigger major
changes in transcription, however, it was the infection with B.
cinerea which greatly impacts the experiment (Fig 3a). Moreover,
whereas separation can be observed between Mock- and B.
cinerea -infected replicates at 9 and 12 hpi, no obvious differences
could be seen at the early time point of 6 hpi.
Genes with similar expression profiles were grouped, resulting in the
identification of 1,722 differentially-expressed genes (DEGs) across all
three treatments and time points. Hierarchical clustering separated the
genes into four crude groups when compared to the Water + Mock treatment
at the first time point (6 hpi, Fig 3b): Cluster i consists of
genes that were repressed by B. cinerea infection; clusterii represents genes induced by chitosan treatment only; clusteriii includes genes repressed by B. cinerea infection and
by treatment of chitosan at the later time points; cluster ivconsists of genes induced by B. cinerea infection and by
treatment of chitosan only (Fig 3b). Overall patterns aligned with the
previous finding that infection with B. cinerea had a
large-scale, more extensive and differential response on tomato
transcription compared to treatment with chitosan (Fig 3a). Moreover,
the analysis demonstrates that application of chitosan results in a
higher number of genes repressed than induced, with the exception of
some highly induced genes in cluster iv . Distinct differences
were evident between treatment with chitosan compared to infection withB. cinerea , e.g. a large group of genes in cluster ivdifferentially induced by B. cinerea at 9 and 12 h, as well as a
large group of genes repressed by the pathogen in cluster i . This
indicates that chitosan works as a priming agent that does not directly
trigger major effects in gene transcription.
To study the different signalling pathways and specific genes
responsible for priming of chitosan against B. cinerea , a two-way
ANOVA identified 8,471 differentially expressed genes (DEGs) among all
three treatments and time points. This global list of DEGs was
subsequently used for focussed pairwise analysis to identify transcripts
changing between treatments at each time-point. Venn diagrams
demonstrates that the effect of chitosan on its own did not trigger
major changes in gene transcription: only 15, 36 and 20 genes were
differentially expressed in Chitosan + Mock vs Water + Mock treatments
at 6, 9 and 12 hpi, respectively (Fig 3c). However, the effect of
chitosan was much more pronounced after plants had been infected withB. cinerea . This combination resulted in the differential
expression of 543, 2,011 and 2,967 genes at 6, 9 and 12 hpi,
respectively, of which 260, 991 and 723 DEGs were induced only in the
chitosan B. cinerea treatment (Fig 3c). In comparison, Water +B. cinerea treatments displayed differential expression of 327,
1,134, and 2,697 genes at 6, 9 and 12 hpi, respectively, of which 70,
116 and 501 DEGs were specific to the Water + B. cinereatreatment (Fig 3c). These results demonstrate that there is a subset of
genes potentially responsible for chitosan-induced priming for a faster
and more robust response against B. cinerea.
To further identify early-acting signalling pathways and genes involved
in chitosan-induced priming, further analyses were performed on genes
corresponding to the 260 probes differentially expressed only in the
Chitosan + B. cinerea treatment at 6 hpi. Gene overrepresentation
analysis was performed to identify biological processes and molecular
functions of enriched genes. For biological processes, pathways such as
response to stimulus, chemical and auxins were overrepresented (Table
1). Moreover, for molecular function, cysteine-type peptidase activity,
transcription factor activity, sequence-specific DNA binding and nucleic
acid binding transcription factor activity were enriched (Table 1).