Throughout their lifetimes, plants are constantly challenged by pathogens. During evolution, a complex and close relationship is developed between plants and pathogens, with infections and defenses being the most crucial relationship. Microbe- (MAMPs) or pathogen-associated molecular patterns (PAMPs) such as fungal chitin, bacterial lipopolysaccharide, flagellin, and peptidoglycan (PGN) are conserved microbe-specific molecules recognized by pattern recognition receptors (PRRs) in the plant innate immune system, known as PAMP-triggered immunity (PTI) (Schwessinger and Zipfel, 2008). Correspondingly, pathogens produce effectors that inhibit the PTI and therefore become more virulent, a state known as effector-triggered immunity (ETI) (Boller and He, 2009). Recent studies have shown that ETI and PTI have a symbiotic crosstalk, which plays a potential interconnected role to create a robust plant defense system (Yuan et al., 2021).

According to the characteristic motifs in the extracellular regions, representative MAMP receptors are classified into distinct subfamilies, such as the leucine-rich repeat (LRR) and lysin motif (LysM) domain–containing receptors (Boutrot and Zipfel, 2017; Saijo et al., 2018). LysM, which contains a highly conserved βααβ secondary structure responsible for the binding of chitin and peptidoglycans (Gust et al., 2012), was first found in Bacillus phage Φ29 lysozyme (Garvey et al., 1986), and then proved to be a ubiquitous protein motif in almost all organisms (Buist et al., 2008). The LysM protein family harbor a variable number of tandem LysM domains (1 to 3). For plant lineage, four types of LysM family proteins are involved: LysM-containing receptor-like kinase (LYK), LysM-containing receptor like protein (LYP), extracellular LysM proteins (LysMe) and nonseretory intracellular proteins (LysMn) (Zhang et al., 2007). LYP and LYK which exist widely in the plant kingdom are relatively well studied among them (Kaku et al., 2006).

CEBiP (chitin elicitor-binding protein) is the first and currently the most clarified LysM protein family. Oryza sativa CEBiP (OsCEBiP) was first identified binding to chitin and subsequently inducing downstream immune responses (Kaku et al., 2006). A similar function of homologous CEBiP was proved in other plants, such as Triticum aestivum CEBiP (TaCEBIP) and Hordeum vulgare CEBiP (HvCEBIP) (Tanaka et al., 2010; Lee et al., 2014; Jian and Liang, 2019). Later, CERK1 (chitin elicitor receptor kinase; also known as LysM-RLK1), the interaction partner of CEBiP, was identified. The knockout of Arabidopsis CERK1 (AtCERK1) completely deprives responses to the chitin elicitor, such as mitogen-activated protein kinase MAPK activation and reactive oxygen species production (Miya et al., 2007, Petutschnig et al., 2010). Oryza sativa CERK1 (OsCERK1) directly interacts with OsCEBiP and forms a receptor complex to cooperatively regulate chitin elicitor signaling (Shimizu et al., 2010). Except for the perception of fungal chitin, the plant LysM protein family was also identified as a bacterial peptidoglycan (PGN) receptor. Arabidopsis LYM1 and LYM3 (also known as LYP1 and LYP3, respectively) are two plasma membrane proteins that directly interact with PGN and mediate the immunity to bacterial infections (Willmann et al., 2011). Some members of the LysM protein family play dual roles in both chitin and PGN perceptions. For example, two rice LysM proteins, OsLYP4 and OsLYP6, were identified as the receptors of both chitin and PGN as responsible for pathogen-induced defense (Liu et al., 2012a; Liu et al., 2013). Besides being the receptor mediating the immune response to fungal and bacterial infections, the plant LysM protein family also functions in plant-microorganism symbiotic interactions. For example, Pisum sativum LYK9 (PsLYK9) can sense the long- and short-chain oligosaccharides of fungi and initiate immune response. On the other hand, PsLYK9 also plays a role in symbiosis development with arbuscular mycorrhizal (AM) fungi (Leppyanen et al., 2021). Similarly, Solanum lycopersicum LYK10 (SlLYK10) and LYK12 (SlLYK12) were also found to be involved in the regulation of AM symbiosis (Buendia et al., 2016; Liao et al., 2018).

The LysM protein can act as a pattern recognition receptor on the cell membrane, transmitting signals to the plant cells and triggering PTI. Moreover, it can also act as an effector to help pathogens penetrate plants, inducing ETI. So far, the most clarified LysM-containing effector is Ecp6, which efficiently binds chitin. Ecp6 was proved to perturb chitin-induced immunity. The probable mechanism is that Ecp6 sequesters chitin fragments to evade the perception by plant immune system (De Jonge et al., 2010; Sánchez-Vallet et al., 2013). Magnaporthe oryzae Slp1 was demonstrated to play a similar role in the inhibition of chitin-triggered immunity (Mentlak et al., 2012). However, unlike Cladosporium fulvum Avr4 and Mycosphaerella graminicola Mg1LysM and Mg3LysM, which can protect fungal hyphae from the hydrolytic activity of chitinases, Ecp6 and Slp1 have no such capacity (De Jonge et al., 2010; Marshall et al., 2011; Mentlak et al., 2012). These studies collectively proved the important role of the deregulation of chitin-triggered immunity by LysM effectors, albeit with varied mechanisms. The suppression of chitin-triggered immunity by LysM effectors has been speculated to be dependent on its ultrahigh affinity for chitin binding, outcompeting the substrate binding by plant chitin receptors such as the LysM protein family (Sánchez-Vallet et al., 2013; Sanchez-Vallet et al., 2015). Fungal LysM effectors are multifunctional proteins that occur in fungal species with different lifestyles, although chitin binding is their most well-known function reported so far. For example, the addition of the purified LysM effector to Trichoderma atroviride inhibits spore germination (Seidl-Seiboth et al., 2013). Our previous study identified 52 candidate fungal effector of Colletotrichum fructicola during its infection on strawberry leaves. Among them, CfLysM2, a LysM effector gene which is homologous to Slp1, was significantly upregulated at the onset of infection. Importantly, the pathogenicity was significantly attenuated after deletion mutation of CfLysM2 (Zhang et al., 2018; Yu et al., 2022).

Strawberry (Fragaria × ananassa Duch.) is one of the most economically important horticultural crops worldwide. However, strawberry plants are prone to many diseases (Hsu et al., 2022). Pathogen-derived injuries or loss due to fungi, bacteria, and so on is a challenge for strawberry producers (Whitaker et al., 2020). Among strawberry diseases, anthracnose disease caused by Colletotrichum fructicola can have devastating effects on strawberry plants (Zhang et al., 2020). The genome of the cultivated octoploid strawberry is one of the most complex plant genomes. It originates from four different diploid ancestors and is an allopolyploid (Edger et al., 2019). The recently published octoploid strawberry genome sequence and transcriptome-wide analysis lay a solid foundation for the identification and analysis of the LysM gene family. At present, 15 FvLysM genes have been identified in wild strawberry (F. vesca) (Liu et al., 2020). Compared with wild strawberry, octoploid strawberry is more susceptible to bacterial and fungal diseases (Martínez Zamora et al., 2008). The LysM proteins are important PRRs involved in plant innate immunity. However, little information is available about the octoploid strawberry. In this study, a genome-wide analysis of LysM genes in the octoploid strawberry (FaLysM genes) was conducted, including classification, physicochemical properties, gene structure, phylogenetic evolution, chromosome location, collinear relationships, transcription factor (TF)-binding sites, and protein model analysis. Nine FaLysM proteins possessed the highly conserved mode, including seven conserved residues (labeled R1–R7) homologous to the OsCEBiP protein in rice. Furthermore, the expression patterns of the FaLysM genes after C. fructicola wild strain (WT) and effector gene CfLysM2 knockout mutant (ΔCfLysM2) infection were profiled. CfLysM2 was previously proved to be highly expressed during infection and contributed to the virulence of C. fructicola (Yu et al., 2022). The distinctive expression profiles of FaLysM genes were observed in strawberry leaves infected with C. fructicola WT and ΔCfLysM2, indicating the complex interaction between C. fructicola and strawberry. These findings provide a basis for further functional characteristics of FaLysM proteins in the octoploid strawberry and insights into the strategies for disease resistance breeding of strawberry.

Proteins containing the LysM motif were proved to be widely distributed in almost all organisms, including eukaryotic and prokaryotic organisms (Buist et al., 2008). In plants, an earlier study confirmed the classical chitin-binding function of one LysM protein family (CEBiP) (Kaku et al., 2006). Since then, proteins containing the LysM motif have been identified in many plants such as Arabidopsis (16), wild strawberry (F. vesca) (n=11), rice (n=15), wheat (n=60), sweet orange (n=9), and Brachypodium distachyon (n=11) (Shiu et al., 2004; Zhang et al., 2009; Zhou et al., 2014; Tombuloglu et al., 2019; Chen et al., 2020; Liu et al., 2020; Li et al., 2021). Previous reports have concluded no meaningful relationship between genome size and the number of genes in plants (Abedi et al., 2021). The observed difference in the number of LysM genes identified in this study may be due to differences in detection criteria. In the present study, 14 LysM proteins were found in octoploid strawberry. The identified FaLysM proteins were categorized into LYK and LYP groups. These proteins were unevenly distributed on 10 chromosomes in the octoploid genome. It should be noted that previous studies identified two LysM proteins containing only one LysM domain in wild strawberry and defined as LysMn proteins (Liu et al., 2020). The LysMn proteins were not identified in our study, indicating the LysMn genes were probably lost or missed in the assembled genome, which may explain the relative disease resistance of wild strawberry to some extent.

Gene duplication events lead to increases in the number of plant genes, which drive genome evolution, in which the creation of new genes is often driven by selective pressure (Murat et al., 2012; Huang et al., 2022). The Ka/Ks ratio is an important index for evaluating evolution across or within the same species (Sironi et al., 2015). In our study, the Ka/Ks ratios of most FaLysM genes were <1, which indicates that its selection pressure was mostly subject to purifying selection during evolution, consistent with the results from banana, wild tomato, and the allotetraploid Brassica napus L (Richards and Rose, 2019; Abedi et al., 2021; Ren et al., 2022). These results suggest that most nonsynonymous substitutions were harmful and were thus eliminated during evolution. The Ka/Ks ratio for one paired gene (FaLysM4/FaLysM6) was >1, indicating positive selection, probably resulting in multiple functions as a result of mutations during their evolution.

Owing to the high purifying selection in the LysM gene family, the importance of the functional role of FaLysM genes has been determined. The phylogenetic tree showed that FaLysM proteins have a close relationship with their counterparts, indicating that the phylogenetic distribution of FaLysM protein is associated with their motif contents. All members of the FaLysM protein family contain 3 common motifs, motifs 1, 5, and 8. Most FaLysM proteins contain motifs 2, 3, 4, and 9. The difference between the members of this subfamily was related to (1) motif 6 being exclusively found in the group of LYPs (FaLysM2, FaLysM5, FaLysM7, FaLysM8, and FaLysM10); (2) motif 7 being exclusively fond in the group of LYKs (FaLysM1, FaLysM11, FaLysM12, FaLysM13, and FaLysM14); (3) motif 10 being exclusively found in the group of LYKs (FaLysM11, FaLysM12, FaLysM13, and FaLysM14); (4) motif 11 being in three types of LYKs (FaLysM1, FaLysM11, and FaLysM13); and (5) motifs 12 and 13 being in three types of LYPs (FaLysM3, FaLysM4, and FaLysM6). These results are completely consistent with the results of the phylogenetic tree. The prediction of the subcellular localization results showed 3 LYPs, namely FaLysM3, FaLysM4, and FaLysM6, all containing up to 10 transmembrane domains, which suggests the possibility that they may act as surface-localized receptors recognizing evading pathogens, consistent with the previous study that showed that CEBiP is a plasma membrane-localized glycoprotein binding to chitin and subsequently inducing downstream immune responses (Kaku et al., 2006).

The phylogenetic analysis of LysM proteins from different plants can provide insights into their potential roles. Chitin elicitor receptor kinase 1 of Arabidopsis (AtCERK1 and AtLYK1), as a critical cell surface receptor, directly binds chitin through the LysM ectodomain to initiate immune responses (Liu et al., 2012b). Overexpression of AtLYK2 enhanced tolerance to fungus and increased the expression levels of defense-related genes during infection (Giovannoni et al., 2021). In our study, a relatively close relationship was observed between AtLYK1 and FaLysM12/FaLysM14, and between AtLYK2 and FaLysM1/FaLysM11/FaLysM13, which suggests that they may also mediate chitin signaling processes. AtLYP1 and AtLYP3 directly interact with PGN and mediate the immunity to bacterial infection (Willmann et al., 2011). From the phylogenetic tree, FaLysM3/FaLysM4/FaLysM6 and AtLYP1 are in the same clade, while other FaLysM proteins and AtLYP3 are in the same clade, which suggests their possible roles in PGN-mediated immunity to bacterial infection. LYKs formed many distinct clades constructed by branches with a single gene, compared with LYPs, possibly indicating that LYKs have undergone faster evolution and maintained relatively more diversity in sequence than LYPs. This phenomenon could also be observed in the NLR family, of which the CNL family is highly diverged compared with the TNL family (Wan et al., 2013; Alamery et al., 2017).

The TFs play an important role in response to environmental stresses by binding to the promoters of the target genes and regulating their expressions (Lindemose et al., 2013). The TF-binding site analysis results suggested that FaLysM genes were involved in various processes during growth and development. As expected, most TFs were associated with plant resistance against biotic stresses, such as NAC, WRKY, ERF, C2H2, bZIP, bHLH, LBD, MYB, and G2-like.

The protein modeling analysis of FaLysM proteins provided insights into their regulation of pathogen-plant interactions. As the first identified chitin oligosaccharide receptor in plants, OsCEBiP is a typical LysM domain-containing protein with two LysMs in the extracellular domain at the N terminus and a transmembrane domain at the C terminus (Kaku et al., 2006). The crystal structures of a free ectodomain of OsCEBiP (OsCEBiP-ECD) in complex with the chitin tetramer (NAG)4 demonstrated that three LysM domains pack tightly against each other, and only the LysM2 domain binds to chitin (Liu et al., 2016). As demonstrated in both plants and fungi, various LysM domain-containing proteins are known to interact with chitin (De Jonge et al., 2010; Sánchez-Vallet et al., 2013; Sanchez-Vallet et al., 2015). A highly conserved mode, including seven conserved residues from the LysM proteins of several plants and fungi, was reported for chitin recognition (Liu et al., 2016). In this study, this mode, especially the several critical residues such as I150 (R6), was also observed in as many as seven FaLysM proteins, demonstrating the potential role of FaLysM proteins in chitin recognition.

PAMPs are defined as highly conserved microbe-specific molecules recognized by PRRs and therefore have an essential function in plant survival or fitness (Jones and Dangl, 2006; Yuan et al., 2021). The expression profiles of these PRR genes provide valuable information on their functions in anti-pathogen plant immunity. Our previous transcriptomic data provides a picture of how strawberry mobilized plant immune system in response to C. fructicola infection, including the differential regulation of several PRR genes (Zhang et al., 2018). In this study, we reanalyzed the RNA-Seq data and focused on FaLysM genes to obtain their expression profiles in response to C. fructicola infection. The results showed the differential regulation of different FaLysM genes in response to C. fructicola infection, implying the complex interaction between C. fructicola and strawberry.

Some PRR genes such as FaLysM4, FaLysM6, FaLysM11, and FaLysM13 have peak expression levels at the late stage of infection (96 hpi), but their early expression levels are not significantly affected, which indicates that although these FaLysM genes were ultimately upregulated at the stage when the leaves developed visible anthracnose symptoms, they appeared to be not activated initially after C. fructicola infection. Another notable example is the significant downregulation of FaLysM1 at 24 hpi upon the onset of C. fructicola infection. Except for these situations, the dominant expression profile is the upregulation of FaLysM genes such as FaLysM3, FaLysM9, and FaLysM10 at the early stage of infection, which indicates that these PRR genes were rapidly stimulated after infection. However, these PRRs, together with some other PRR genes such as FaLysM2, FaLysM5, FaLysM7, FaLysM8, FaLysM12, and FaLysM14, were downregulated at the late stage of infection. A recent study also showed the elevated expression levels of candidate LysM genes at 6 to 24 hpi, followed by a decrease at 48 hpi, in Coffea arabica infected with Hemileia vastatrix (Santos et al., 2022). These results describe that most PRR genes exhibit an initial stimulus with subsequent suppression upon fungus infection. Collectively, the distinctive expression profiles of the FaLysM genes are reminiscent of the complex and competitive relationship between the CfLysM2 and FaLysM genes, just like the similar expression profiles of other PRRs in our previous results, such as leucine-rich repeat receptor-like protein kinases (LRR-RLKs) (Zhang et al., 2018).

Various studies have demonstrated the interference of chitin-mediated plant immunity by LysM effectors elicited by different fungi (De Jonge and Thomma, 2009; Marshall et al., 2011; Mentlak et al., 2012). A typical example is the sequestration of chitin by Ecp6, a secreted LysM effector of the fungus Cladosporium fulvum, to perturb chitin-induced tomato immunity (De Jonge et al., 2010). A similar function was also proved for Magnaporthe oryzae Slp1 and Mycosphaerella graminicola Mg3LysM (Marshall et al., 2011; Mentlak et al., 2012). The inhibition of PRR expression may be associated with the suppression of PTI signaling (Santos et al., 2022). From our transcriptome data, we chose the significantly upregulated LysM effector gene CfLysM2 during C. fructicola infection, to generate the knockout mutant CfLysM2. By comparing the expression profiles of the FaLysM genes in response to both WT and ΔCfLysM2, we conclude that the inhibition of most FaLysM gene expressions by CfLysM2 is reminiscent of the possible competition between C. fructicola LysM effectors and FaLysM proteins.

In this study, 14 FaLysM protein members were identified in octoploid strawberry, including 5 LYKs and 9 LYPs. The protein model analysis results showed that nine FaLysM proteins contain a conserved mode of chitin binding, which suggests their potential roles in pathogen perception and plant immunity. The distinct expression profiles of the FaLysM genes upon the onset of C. fructicola infection suggest the complex interaction between C. fructicola and strawberry. By comparing the expression profiles of the FaLysM genes upon the onset of C. fructicola WT and ΔCfLysM2 infections, we found that most FaLysM gene expressions are broadly inhibited by CfLysM2 infection. These findings provide fundamental information on the strawberry LysM proteins and their interactions with C. fructicola, which promotes in-depth investigations on the interaction between C. fructicola and strawberry and on resistance breeding of strawberry.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The study was conceived by LZ, HA and XZ. LZ prepared the C. fructicola isolate and plant materials and performed the experiments. SL performed the bioinformatics analysis. LZ and XF performed the experiments of infection treatment, RNA Extraction, and qRT-PCR Analysis. LZ, HA and XZ prepared the manuscript. All authors contributed to the article and approved the submitted version.