Genome-wide distribution and characterization of LysM genes in various plant species
LysMs, carbohydrate-binding modules with a length of approximately 40 amino acids, can bind to N-acetylglucosamine (GlcNAc)-containing glycans, such as peptidoglycan, chitin, and chitin-like compounds [44]. These genes therefore usually act as PRRs to detect PAMPs of invading pathogens, and activate defense genes and plant innate immunity. Based on the genomes of several plants, systematic genome-wide investigation of LysM genes has been carried out in these species.
LysM genes are distributed unevenly in the chromosomes in a variety of species [45]. For example, in Arabidopsis, 14 LysM genes, 5 Lyks, 3 Lyps, 3 LysMes, and 3 LysMns, are distributed throughout all five of the chromosomes. In Glycine max, 47 LysM genes, comprising 21 Lyks, 4 Lyps, 16 LysMes, and 6 LysMns, are also positioned on all chromosomes, expect chromosome 12 (https://phytozome.jgi.doe.gov/pz/portal.html). However, in rice, 20 LysM genes, comprising 6 Lyks, 6 Lyps, 4 LysMes, and 4 LysMns are only positioned on 8 of 12 chromosomes, and in P. trichocarpa, 35 LysM genes, comprising 11 Lyks, 7 Lyps, 10 LysMes, and 7 LysMns, are distributed in 12 of 19 chromosomes. The differences in the distribution of LysM genes between species might be related to the individual defense characteristics of each species that developed during the evolutionary process, and interestingly, many duplicated LysM genes also display different expression patterns [10, 45]. Here, we first systematically identified 29, 30, 60, and 56 LysM genes in four sequenced cotton species; the diploid cottons G. raimondii and G. arboreum, and the tetraploid cottons, G. hirsutum acc. TM-1 and G. barbadense acc. 3–79, respectively. The 29 LysM genes in G. raimondii were anchored to all 12 chromosomes, except Chr. D7, implying that the LysM genes are widely distributed in the Gossypium genome. From an evolutionary point of view, we can consider that one member of the LysM gene family in the diploid species G. raimondii corresponds to one homologous gene in G. arboreum and two homologs from the A and D subgenomes in tetraploid G. hirsutum acc. TM-1 and G. barbadense acc. 3–79. We found that 14 members of the LysM gene family had such a correspondence in the four sequenced cotton species, indicating that the A- and D-subgenomes evolved independently after polyploid formation (Additional file 1: Table S1). The other inconsistencies may result from chromosome segmental or tandem duplication events during the evolution of different cotton species, the sequence quality and type of sequencing methods used in different cotton species, or assembly error in partial chromosomal regions. This requires further investigation.
The secretory pathways, signal peptides and transmembrane domains of proteins are particularly crucial to cellular function during defense against both biotic and abiotic stresses [46]. In this study, characteristics of the LysM genes, including their signal peptides (SPs), subcellular localization, and transmembrane domains were investigated in G. raimondii (Additional file 3: Table S2). We found that SPs existed in 15 LysM proteins, suggesting that the proteins are synthesized as pre-proteins, are subsequently cleaved at the signal peptide site to form a mature protein, and function by targeting the general secretary pathway. In addition, 12 LysM proteins, comprising all the GrLyks, GrLyp1, GrLysMe3, GrLysMe9, and GrLysMn7, possessed transmembrane domains; indicating that they have multiple complex functions. In addition to containing transmembrane domains and being involved in secretory pathways, eight LysM proteins, Lyp1–5, Lyk7, Lyk8, and Lyp1, also possessed signal peptides, indicating that they play important roles in the defense against a variety of stresses in cotton.
LysM genes show multiple expression patterns
Previous reports have shown that the expression of plant LysM genes can be both constitutive and induced [10, 11, 47, 48]. Most LysM kinase genes in Glycine max are predominantly expressed in the roots, and orthologous genes have similar tissue expression patterns [10]. In the present study, we systematically analyzed the expression patterns of LysM genes in cotton. Using transcriptome data from G. hirsutum acc. TM-1 vegetative and reproductive organs, a total of 50 LysM genes were found to have diverse developmental and spatial regulation patterns (Fig. 2), and most genes showed both diverse and overlapping expression patterns in various tissues and organs, suggesting that these genes have a range of functions but with the identical conserved domains.
There is an increasing volume of data to indicate that LysM-containing proteins can detect several PAMPs, such as the bacterial oligosaccharide, peptidoglycan, and fungal chitin. Upon detection of PAMPs, these proteins then activate a wide range of physiological responses, including the production of SA, JA, ET, and ROS, as well as mitogen activated protein kinase (MAPK) phosphorylation, calcium influx, and the expression of defense-related genes [18, 49]. In A. thaliana, AtCERK1 was shown to be a key chitin receptor, and mediates chitin-induced signaling through homodimerization and phosphorylation [12, 14], and in rice, CEBiP associates with chitin-elicitor receptor kinase 1 (OsCERK1) to mediate MAMP-triggered immunity (MTI) in response to chitin [17, 50]. To better validate the roles of cotton LysM genes in chitin recognition and chitin signal transduction, we analyzed the expression patterns of 20 highly expressed LysM genes in different Hai7124 tissues following treatment with insoluble crab shell chitin and soluble chitin fragment N-acetylchitohexaose. Sixteen genes were significantly upregulated in response to these two PAMPs at different time points, suggesting that LysM proteins play important roles in chitin recognition (Fig. 3). Further, we found that 12 LysM genes, 4 Lyks, 3 Lyps, 1 LysMe, and 4 LysMns, had high expression levels in Hai7124 after inoculation with V. dahliae strain V991, reaching peak expression levels at different time points (Fig. 4). Lyp1, Lyk7, LysMe3, and LysMn6 were significantly induced following treatment with both chitin signals and V. dahliae. These findings imply that the LysM genes play critical roles in fungal perception and function in cotton defense mechanisms against V. dahliae.
The G. hirsutum and G. barbadense species probably originated from a single hybridization event between A- and D- diploid species, however, the two have very different agronomic and fiber quality characteristics. For example, most of modern G. barbadense cultivars are resistance to V. dahliae, however, G. hirsutum cultivars are not. As described above, we found that Lyp1, Lyk7 and LysMe3 were significantly upregulated in cotton roots at different time points after inoculation with V. dahliae, and had higher expression levels in G. barbadense cv. Hai7124 than G. hirsutum cv. Junmian 1, indicating that these genes act as positive regulators in plant resistance to V. dahliae.
LysM genes function in the defense against biotic stresses
LysM genes mainly function in recognizing chitin elicitor signals and activating plant immune responses. This has been investigated in various plant species, such as Arabidopsis, wheat, rice, and tomato [15, 17, 47, 51]. In Arabidopsis, CERK1, a LysM receptor kinase, responds to chitin elicitors resulting in MAPK activation and ROS generation [14]. Like CERK1, LYK4 and LYK5 have been localized to the plasma membrane, and are involved in chitin recognition, chitin signal transduction and plant innate immunity [15, 16]. LYM1 and LYM3, two plasma membrane proteins, physically interact with peptidoglycans and mediate immunity to bacterial infection [33]. In rice, a plasma membrane glycoprotein, OsCEBiP, and a receptor kinase, OsCERK1, act as critical components of chitin signaling recognition pathways [17]. Similarly, LYP4 and LYP6 are promiscuous PRRs for PGN and chitin recognition, and are involved in ROS generation, defense gene activation, and callose deposition in rice [18]. In Medicago, LYK3 is proposed to function as the entry receptor in rhizobial nodulation factor signaling and specifically controls responses to infection [48]. Taken together, these data show that LysMs are important in the recognition of PGN and chitin signals, and the activation of plant immune responses to biotic stresses.
In this study, cotton Lyp1, Lyk7, LysMe3, and LysMn6 genes were upregulated at different time points after V. dahliae treatment. We observed that Lyp1-, Lyk7, and LysMe3-silenced G. barbadense cv. Hai7124 plants had more-severe disease symptoms than the TRV: 00 control and LysMn6- silenced plants after V. dahliae infection (Figs. 5b and 6b). In addition, statistical analysis suggested that the percentage of diseased leaves in Lyp1-, Lyk7-, and LysMe3-silenced plants was significantly higher than in control plants (Fig. 5c, Additional file 7: Table S3; Fig. 6c, Additional file 8: Table S4). These findings imply that Lyp1, Lyk7, and LysMe3, which have transmembrane domains, function as important chitin receptors, allowing them to recognize V. dahliae and induce cotton immunity.
Lyp1, Lyk7, and LysMe3 contribute to V. dahliae resistance through the activation of plant innate immune responses
Chitin, a major component of the fungal cell wall, is a typical PAMP and is recognized by PRRs, which activate PTI. PTI is characterized by a wide range of physiological responses, including the production of ROS, calcium influx, and the expression of defense-related genes [4]. In addition, chitin signaling pathways appear to be independent, and different PAMPs might activate a common downstream pathway to induce pathogen resistance [1, 52, 53]. An increasing volume of data further demonstrates that plasma membrane receptors recognize chitin signals and activate downstream SA, JA, ET, and ROS pathways [3, 18, 54].
Chitin binding sites have been identified in the plasma membrane of several plants [5,6,7,8]. Here, we analyzed the subcellular location of Lyp1, Lyk7, and LysMe3, and obtained the direct evidence that these are membrane-anchored proteins (Fig. 7). To determine whether Lyp1, Lyk7, and LysMe3 affect downstream SA, JA, ET, and ROS production, we examined the expression of Lyp1, Lyk7, and LysMe3 after SA, JA, ET, and H2O2 treatments. Compared to the control, all three genes were significantly induced after SA, JA, and H2O2 treatment (Fig. 8a). We also examined the expression of several important genes in the SA pathway, GbEDS1, GbPAD4, GbEDS5, and GbSID2 [35, 36], the JA pathway, GbAOS, GbOPR3, GbMYC2, and GbJAZ1 [37], and the ROS pathway GbRBOH, GbCAT1, GbPOD, and GbSOD [38, 39] in VIGS plants. These genes were significantly down-regulated in the Lyp1-, Lyk7-, and LysMe3-silenced cotton plants compared to the control (Fig. 8b), indicating that the SA, JA, and ROS pathways were perturbed in Lyp1-, Lyk7-, and LysMe3-silenced plants.
PR genes play important roles in signal recognition and plant immunity. For example, in tobacco, PR1 is involved in the stress response and is associated with resistance to oomycete pathogens [40]. PR4 encodes chitinase, which is an endogenous plant defense enzyme that generates signaling molecules (elicitors) for the induction of further defenses [41]. In A. thaliana, PR5 is thought to regulate SA biosynthesis and lead to the accumulation of high levels of camalexin in order to protect the plant against pathogen infection [42]. In addition, PR10 is suggested to increase pepper’s resistance to the oomycete pathogen, Hyaloperonospora arabidopsidis [43]. In the present study, we examined the expression of several defense-related marker genes, PR1, PR4, PR5, and PR10, in Lyp1-, Lyk7-, and LysMe3-silenced VIGS plants. The four PR genes were significantly upregulated in control plants after V. dahliae infection (Fig. 9a); but were all significantly down-regulation in the Lyp1-, Lyk7-, or LysMe3-silenced plants compared to the control, suggesting that defense-related processes were restrained in the VIGS plants, leading to moderate susceptibility to fungal pathogens such as V. dahliae. When challenged by pathogens, plant levels of the signaling compounds, SA, JA, ET, and ROS change, PR gene expression is induced and plant resistance to infection is enhanced [55, 56]. In our study, the expression of PR1, PR4, PR5, and PR10 was significantly increased after SA, JA, or ROS treatment in cotton plants, but was significantly lower in the Lyp1-, Lyk7-, and LysMe3- silenced cotton plants (Fig. 9b, c). Taken together, these findings suggest that cotton LysM-containing proteins are involved in activating downstream defense processes to enhance resistance to V. dahliae.
Recent studies have shown that common mechanisms for receptor activation involve receptor homodimerization or oligomerization and subsequent phosphorylation, and that the LysM receptors might function as protein complexes [16]. In Arabidopsis, AtFLS2 was associated with AtBAK1 upon flagellin treatment, which initiated cellular defense signaling [57, 58]. In addition, the AtLYK5-AtCERK1 interaction is chitin dependent, yet AtLYK4 can interact with AtCERK1 independently of the presence of chitin [16]. Here, we confirmed that the three LysM-containing proteins, Lyp1, Lyk7, and LysMe3, act as plasma membrane receptors in cotton and recognize chitin signals, activate downstream SA, JA, and ROS pathways, induce PR gene expression, and enhance resistance to V. dahliae (Fig. 10). However, the functional characteristics of Lyp1, Lyk7, and LysMe3 in the resistance network remain to be clarified. Future investigations into the effect of the three LysMs on specific defense-related genes and their involvement in the defense network will be meaningful, not only to improve our understanding of the molecular mechanisms of LysM-chitin interactions, but also for developing fungal-resistant cultivars through breeding programs for cotton and other crops.