INTRODUCTION
Cotton (Gossypium spp.) cultivation has a major impact in human
society since it is widely used as a source of fiber and seed oil.
Verticillium wilt disease caused by the soil-borne vascular diseaseVerticillium dahliae is one of the major threats limiting cotton
productivity. Diseased cotton is characterized by wilting, stunting,
chlorosis, vascular discoloration, early senescence (Xu et al., 2013).
Strains of V. dahliae isolated from cotton have been
characterized as causing a defoliating or non-defoliating phenotype, and
the genetic basis for the defoliating phenotype was recently established
(Zhang et al. 2019). In either case, there are few resistant germplasm
sources or efficient management measures available to control this
pathogen, especially post-infection. Verticillium dahliae has a
very broad host range, infecting over 200 plant species, and survives
for years in the soil, precluding crop rotation as a strategy for
disease control (Klosterman et al. 2009).
The
evolutionary arms race between plants and pathogens has prompted the
development of innate immune responses. One of these immune responses is
known as MAMP (microbe-associated molecular pattern)-triggered immunity
(MTI), and this response serves as the first barrier to guard against
the invasion of pathogens (Chisholm et al., 2006). Another immune
response is known as effector-triggered immunity (ETI), which is carried
out by dominant resistance (R) proteins by recognizing pathogen effector
proteins directly or indirectly, to trigger gene-for-gene resistance
(Jones and Dangl, 2006). The canonical R proteins contain a nucleotide
binding site (NBS) and leucine-rich repeat (LRR) domains. The core
nucleotide binding domain in NBS-LRR proteins is known as the NB-ARC
domain since it is well studied in APAF-1 (apoptotic protease-activating
factor-1), R proteins, and CED-4 (Caenorhabditis elegans death-4
protein) (van der Biezen and Jones, 1998). In plants, NBS-LRR proteins
mediate pathogen-specific effector triggered immunity, and the genes
that encode them are widely used as markers in plant breeding to
generate disease resistance. A drawback of R -mediated resistance
is that it occurs at the expense of fitness (Tian et al., 2003), which
suggests that the expression of an R gene must be highly
regulated, and strictly inactivated in the absence of a pathogen.
Reactive oxygen species (ROS) also play multiple roles in MTI and ETI
defense. For example, ROS can function as secondary messengers directly
or indirectly to activate the expression of defense-related genes and
induce programmed cell death during the hypersensitive response (HR)
(Mittler, 2017; Mittler et al., 2011). However, excessive ROS adversely
affects many cellular functions by causing oxidative damage to DNA, RNA,
proteins and membranes (Apel and Hirt, 2004). Plants have evolved many
antioxidative systems to eliminate ROS, including enzymatic and
nonenzymatic mechanisms. Enzymatic ROS scavenging mechanisms in plants
include superoxide dismutase (SOD), glutathione peroxidase (GPX),
ascorbate peroxidase (APX), and catalase (CAT). SODs act as the first
line of defense against ROS. There are three types of SODs in plants
based on different metal ligands involved copper/zinc SOD (Cu/Zn–SOD,
also known as CSD), manganese SOD (Mn–SOD) and iron SOD (Fe–SOD) (Guan
et al.,
2013).
There are three
CSD
isozymes which are localized in different cellular compartments inArabidopsis : cytosolic CSD1, chloroplastic CSD2 and peroxisomeic
CSD3 (Huang et al., 2011). In Arabidopsis , there is a copper
chaperone for superoxide dismutase (CCS, which delivers copper to the
CSD) to activate all three CSD isozymes activities (Huang et al., 2011).
Overexpression of CSD1 and CSD2 enhances the tolerance of
transgenic plants to UV and high light treatment, salt and heavy metal
stresses (Leng et al., 2017; Sunkar et al., 2006). SODs have also been
reported to play roles in the hypersensitive response (HR) during
cotton-Xanthomonas campestris interaction and
barley-Blumeria graminis interaction (Voloudakis et al., 2006; Xu
et al., 2014b).
MicroRNAs (miRNAs) are approximately 21 or 22 nucleotide (nt) long
non-coding RNAs that play essential roles in gene silencing by targeting
mRNA for cleavage, or by translational repression in both plants and
animals (Reinhart et al., 2000; Reinhart et al., 2002). Primary miRNAs
(pri-miRNAs) are transcribed by RNA polymerase II but contain an
imperfect stem-loop or hairpin structure (Voinnet, 2009; Xie et al.,
2015). miRNAs are released from their pri-miRNAs by RNase III-like
Dicer-like enzymes in plants (Margis et al., 2006), and then associate
with argonaute (AGO) protein (Mallory and Vaucheret, 2010) to inhibit
gene expression at transcriptional gene silencing (TGS) or
post-transcriptional gene silencing (PTGS) levels (Bologna and Voinnet,
2014). Hundreds of miRNAs have now been discovered through
deep-sequencing and genetic approaches (Meyers et al., 2006), and they
have been shown to play vital roles in plant development (Couzigou and
Combier, 2016; Guo et al., 2017; Huang et al., 2017; Zhang et al., 2017)
and responses to biotic and abiotic stresses (Deng et al., 2018; Ding et
al., 2017; Kumar, 2014; Li et al., 2017).
Intriguingly, miRNAs are considered as important regulators of Rgene expression. For example, two miRNAs, nta-miR6019 and nta-miR6020,
were reported to guide the cleavage of the tobacco mosaic virus (TMV)
resistance gene, N , which is a toll and interleukin-1
receptor-NBS-LRR immune receptor (Li et al., 2012). Furthermore,
miR482/2118 is another well-known miRNA that targets the NBS-LRRresistance genes (Shivaprasad et al., 2012). Therefore, miRNA-mediated
repression of NBS-LRR genes is an efficient mechanism for plants
to balance the trade-off between growth and defense. The miRNA known
specifically as miR398 has been reported to play a role in responses to
various abiotic stresses by modulating the expression of its target
genes (Zhu et al., 2011; Wang et al., 2016). miR398b was the first miRNA
reported to be down-regulated in response to biotic stress (P.
syringae ) in Arabidopsis (Jagadeeswaran et al., 2009; Li et al.,
2010). In our previous work, we also found that miR398b plays a role in
temperature stress through miRNA and degradome sequencing (Wang et al.,
2016). To date, four targets of miR398 have been reported through
computational prediction and sequence analysis: CSD1 ,CSD2 , CCS and COX-5b (a subunit of the
mitochondrial cytochrome c oxidase) in Arabidopsis (Beauclair et
al., 2010; Jones-Rhoades and Bartel, 2004). Some studies also show that
miR398 negatively regulates the PTI response and resistance to
pathogenic bacteria (Li et al., 2010). However, whether miR398 can
participate in the ETI response and resistance to fungal pathogensV. dahliae is not known. Here, we investigated the potential role
of miR398b in cotton-V. dahliae interaction. The results indicate
that miR398b can target both NBS-LRR genes and CSD family genes
to suppress the resistance of cotton
to V. dahliae .