DISCUSSION
Previously we identified differentially expressed miRNAs including
miR8746, miR482, and miR398b, which were putatively involved in
targeting NBS-LRR disease resistance transcripts (Wang et al.,
2016). In this current study, we demonstrated that overexpressing
miR398b made plants more susceptible to V. dahliae in cotton
(Figure 2). In support of these finds, miR398 has been reported to
negatively regulate MAMP-triggered immunity (Li et al., 2010) and the
expression levels of miR398 were down-regulated upon inoculation of
plants with incompatible strains DC3000 (avrRpm1) and DC3000 (avrRpt2),
but not the compatible strain DC3000 (Jagadeeswaran et al., 2009). This
implicates involvement of miR398 in both MTI and ETI. In our study, we
found that miR398b can target two NBS-LRR genes
(Gh_D05G3257 and Gh_A04G0380 ) and the target site of
the genes is in the internal region, a region not completely conserved
in NBS-LRR genes. No identical target sequences were found in the
homologous genes in Oryza sativa , Arabidopsis thaliana ,Zea maize , Populus trichocarpa and Theobroma cacao .Gh_D05G3257 and Gh_A04G0380 may be unique target genes
of miR398b at the post-transcriptional level and contribute to cotton
resistance against V. dahliae (Figure 3, Figure 4, Figure 5,
Table S1). To our knowledge, this is the first report that
miR398b
can target plant NBS-LRR genes to regulate defense responses to a
pathogen.
ROS is a general term for oxygen-derived metabolic intermediates or
radicals including superoxide (O2∙-), hydrogen peroxide (H2O2), hydroxyl
radical (OH∙) and singlet oxygen (1O2) (Apel and Hirt, 2004).
ROS-generation and -scavenging pathways play an important role both in
pathogen virulence and resistance by the host.
The
ROS burst is reported to be essential for penetration peg formation byV. dahliae , and therefore is indispensable for virulence and
colonization by this fungus (Zhao et al., 2016). Moreover, excess ROS
produced during plant-pathogen interactions facilitates plant infection
and colonization by necrotrophic pathogens (Chung, 2012; Govrin and
Levine, 2000). In the host, rapid ROS accumulation also occurs during
the cotton-V. dahliae interaction but it is quickly scavenged to
maintain homeostasis. Thioredoxin GbNRX1, an important apoplastic
ROS-scavenger, plays a positive role in the cotton response to V.
dahliae (Li et al., 2016) and silencing of anthocyanidin synthase
(GbANS ) in cotton increased H2O2production and cell death around the invasion sites, which in turn leads
to increased V. dahliae infection (Long et al., 2018). These
results indicate that ROS elimination after the initial burst may be
functionally important in cotton resistance to V. dahliae .
Among other players involved in ROS
metabolism, superoxide dismutases (SODs) are found in all kingdoms of
life and act as the first line of defense against ROS, dismutating
superoxide to H2O2 (Miller, 2012). In
this study, we found that overexpression of miR398b boosted more
H2O2 production when treated with NLP1
and V. dahliae and therefore accelerates the NLP1-triggered cell
necrosis (Figure 7). By contrast, overexpression of GhrCSD2 ,
which is resistant to miR398b, decreased
H2O2 content (Figure 7B). These results
were strikingly similar to those of recent reports from analyses of rice
upon Magnaporthe oryzae infection (Li et al., 2019).
Additionally, expression levels of GhMSD1 , GhFSD2 andGhRbohB (the gene putatively encoding NADPH oxidase to catalyze
the production of O2∙- ) were
decreased in GhrCSD2- overexpression lines upon NLP treatment, but
increased in miR398b-overexpression lines compared to WT plants.
Meanwhile, transcripts of GhCSD1 , GhCCS andGh_D05G3257 (the other target genes of miR398b) were increased
in GhrCSD2- overexpression lines and decreased in
miR398b-overexpression lines (Figure S6). These results suggest that a
compensatory regulation mechanism may also exist in cotton ROS
homeostasis. However, the phenotypes of miR398- and
GhrCSD2-overexpression plants in cotton upon pathogen were differ from
those in rice. Overexpression of miR398b in cotton impaired cotton
resistance to V. dahliae , while overexpression of miR398b in rice
enhanced resistance to M. oryzae (Li et al., 2019). We propose
that the excessive ROS accumulation in miR398b-overexpressing plants may
damage the cell and facilitate V. dahliae infection (Figure 7),
consistent with results of previous studies (Chung, 2012; Li et al.,
2016).
Taken together, these results suggest that miR398b may function in one
of two different ways to regulate plant immunity. First, the results
indicate involvement of miR398b in ROS homeostasis through cleavingGhCSD1 , GhCSD2 and GhCCS1 , and second, miR398b
suppresses the translation of NBS-LRR genes to control defense
response (Figure S7). An in-depth account of how miR398b expression is
regulated during cotton-V. dahliae interactions will be an
interesting topic for a future study.