Plant diseases occur from the interaction between host plants and pathogens. Based on plant innate immunity theory, the recognition of conserved microbial surface structures termed ‘microorganism-associated molecular patterns’ (PAMPs) by cell surface pattern recognition receptors (PRRs) to elicit pattern-triggered immunity (PTI; Jones and Dangl, 2006). PTI confers full immunity to host non-adapted pathogens and partial immunity to host-adapted pathogens (Wang et al., 2022). To successfully infect host plant, the latter pathogens have evolved ways to evade PTI by secreting effectors into plant cells, which was termed effector triggered susceptibility (ETS). In turn, plants have evolved resistance (R) proteins, which recognize effectors and led to effector triggered immunity (ETI) which was mostly characterized by a hypersensitive response (HR; Jones and Dangl, 2006; Stergiopoulos and de Wit, 2009). These interactions are complex, not only involving in multifaceted recognition of pathogens by the plants but also involving in subtle evasion from the pathogens (Campos et al., 2021). In plants, there are a large repertoire of genes encoding cell surface and intracellular immune receptors for various danger signals detection to active effective defenses against pathogen infection. However, this also drives pathogens to evolve mechanisms of plant immune recognition evasion to facilitate colonization, such as evasion of PRR-mediated detection though altering, sequestering, degrading or preventing release of MAMPs (Wang et al., 2022).

Lysin motif (LysM) was originally identified as a protein domain in a Bacillus phage lysozyme that degraded bacterial cell walls (Garvey et al., 1986). So far, a large number of LysM-containing proteins were found in eukaryotes including plants and fungi (Buist et al., 2008). As receptors, plant LysM-containing proteins could recognize chitin, a well-known PAMP released from fungal cell walls during infection, to trigger PTI (Miya et al., 2007; Kombrink et al., 2011; Thomma et al., 2011; Sanchez-Vallet et al., 2015). However, many fungal pathogens could secrete LysM-containing effectors similar to plant’s chitin receptors (Hu et al., 2021), sequestering and masking fungi to prevent fungal cell wall degradation by plant chitinases (Kombrink and Thomma, 2013; Sanchez-Vallet et al., 2015; Volk et al., 2019; Gong et al., 2020), and inhibiting chitin-induced plant immunity by blocking chitin sensing or signaling (Bolton et al., 2008; Jonge et al., 2010; Marshall et al., 2011; Mentlak et al., 2012; Lee et al., 2014; Takahara et al., 2016; Kombrink et al., 2017; Dolfors et al., 2019; Tian et al., 2021). Cladosporium fulvum Ecp6 was a typical LysM protein with three LysM domains and could chelate chitin during infection (Bolton et al., 2008). During infection, Ecp6 sequesters chitin oligosaccharides released from the cell walls of invading hyphae to prevent chitin mediated PTI (Jonge et al., 2010). In contrast to C. fulvum Ecp6, Mycosphaerella graminicola Mg1LysM and Mg3LysM also protected fungal hyphae against plant-derived hydrolytic enzymes, however, only Mg3LysM blocked the elicitation of chitin-induced plant defenses (Marshall et al., 2011). Mg3LysM deletion mutant showed severely impaired ability in leaf colonization and lesion formation, and was nonpathogenic toward the wheats silenced either CERK1 or CEBiP which were required for activation of chitin-induced defenses as the receptor-like proteins in wheat (Marshall et al., 2011; Lee et al., 2014), indicating the importance of Mg3LysM for fungal evasion of PTI through blocking chitin signaling. The LysM-containing effectors, such as Slp1 in Magnaporthe oryzae, ChELP1 and ChELP2 in Colletotrichum higginsianum, Vd2LysM in Verticillium dahliae, RsLysM in Rhizoctonia solani, Mgx1LysM in Zymoseptoria tritici, had been proved to contribute to virulence by disturbing the activation of chitin-triggered immunity (Mentlak et al., 2012; Takahara et al., 2016; Kombrink et al., 2017; Dolfors et al., 2019; Tian et al., 2021). The evolutionary study from 57 endophytes and fungi with diverse lifestyles showed that LysM effectors contributed to fungal lifestyle (Suarez-Fernandez et al., 2021). Additionally, RiSLM, a secreted LysM effector from arbuscular mycorrhiza (AM) fungal species Rhizophagus irregularis, protected fungal cell walls from chitinase by binding to chitin oligosaccharides and effectively interfere with chitin-triggered immune response to subvert chitin-triggered immunity during symbiosis (Gust et al., 2012; Zeng et al., 2020). Similarly, the beneficial fungus Trichoderma viride employed a LysM effector Tal6 to sequester GlcNAc oligomers, thereby interfering with the perception of fungal-derived N-acetylglucosamine by plants surveillance machinery leading to protection of fungal hyphae from the host’s chitinases (Romero-Contreras et al., 2019).

Some evidences had demonstrated that fungal LysMs were not always only involved in manipulation of plant immunity, but also in the self-regulation of fungal growth and development. Trichoderma atrovirideis LysM protein TAL6 was shown to specifically inhibit germination of Trichoderma spp. rather than fungal-plant interactions (Seidl-Seiboth et al., 2013). PeLysM3 was identified from Penicillium expansum and contained one LysM domain, which null mutant exhibited slightly lower rate of radial growth, significantly lower percent of germinating spores and shorter germ tubes (Levin et al., 2017), but Verticillium nonalfalfae LysM protein VnaChtBP deletion had no significant effect on the growth and pathogenicity (Volk et al., 2019). In M. graminicola, the LysM effector Mg1LysM negatively regulated pycnidia formation on wheat, but Mgx1LysM and Mg3LysM positively regulated pycnidia formation (Tian et al., 2021).

In Colletotrichum, two LysM effectors ChELP1 and ChELP2 had been identified in C. higginsianum. Both ChELP1 and ChELP2 were essential for fungal virulence and appressorium-mediated penetration and suppressed the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases (Takahara et al., 2016). C. graminicola also contains two homologs of Ecp6 which had been experimentally proven to interact with chitin and modulate plant immunity (Sanz-Martin et al., 2016). Natural rubber is an irreplaceable important strategic resource and industrial raw material and mainly produced from rubber tree (Hevea brasiliensis). The rubber tree anthracnose, caused by filamentous fungus Colletotrichum gloeosporioides, resulted in serious loss of natural rubber production worldwide (Liu et al., 2018; Wang et al., 2018). However, little is known about the pathogenesis of C. gloeosporioides toward rubber tree. To elucidate the pathogenic mechanism of C. gloeosporioides, the candidate genes encoding effectors were predicted in the genome of C. gloeosporioides and one of candidate effector containing two LysM was named Cg2LysM. In this study, the contribution of Cg2LysM in fungal virulence and development was investigated. These results enriched our understanding of the molecular pathogenesis mechanisms of C. gloeosporioides on rubber tree.

Multiple LysM-containing proteins were found in both prokaryotes and eukaryotes. Many fungal pathogens encoded effectors containing LysM domain (de Jonge and Thomma, 2009). Based on evolutionary analyses, LysM domains were classified into two groups: fungal-specific group and fungal/bacterial group (Akcapinar et al., 2015). The LysM motif of fungal/bacterial group consisted of about 40 amino acids with none or only one cysteine, e.g., those of Cladosporium fulvum Ecp6, Mycosphaerella graminicola Mg3LysM and Magnaporthe oryzae Slp1(Bolton et al., 2008; Marshall et al., 2011; Mentlak et al., 2012); the LysM domain of fungal specific group consisted of 53 amino acids with four conserved cysteines (positions 5, 17, 42 and 52) and an N in a WNP (positions 35–37), e.g., those of Tricho derma atroviride Tal6 (Seidl-Seiboth et al., 2013). Based on their overall domain architecture, fungal LysM proteins were classified into five types. Of them, the members of Type A had a signal peptide and did not contain any recognizable motif other than a varying number of LysMs, which were referred to as “LysM effectors” and might play roles in the infection process; However, Type B-E contained not only one or more LysM but also other recognizable conserved domains such as Cys-rich chitin-binding domain, enzymatic domain, CyanoVirin-N homology domain, chitin recognition domain and so on (de Jonge and Thomma, 2009). In this study, Cg2LysM had two LysM motifs of about 40-aa (46–87aa and 115–158aa), the former LysM motif containing a cysteine (position at 65aa) and the latter without cysteine (Supplementary Figure S1A), which was consist with the features of fungal/bacterial group of LysM-containing proteins. Cg2LysM had only a recognizable N terminal signal peptide (1–27aa) in addition to two LysM motifs (Supplementary Figure S1A), and Cg2LysM was clustered with type I of known LysM proteins in phylogenetic tree, indicating that Cg2LysM was a A-type LysM effector protein.

As pathogenic factor, some LysM effector proteins contributed to fungal virulence and regulation of fungal growth and development. In Cladosporium fulvum, RNAi-mediated silencing of Ecp6 resulted in a clearly delayed progression of disease on tomato plants, and heterologous expression of Ecp6 in Fusarium oxysporum f. sp. lycopersici enhanced virulence on tomato (Bolton et al., 2008). In Mycosphaerella graminicola, Mg3LysM deletion mutant showed only weak virulence toward wheat leaves companying with a complete lack of necrotic lesions bearing pycnidia and a dramatically reduced fungal biomass (Marshall et al., 2011). In Magnaporthe oryzae, deletion of a LysM effector protein SLP1 significantly reduced the ability to cause disease, which was associated with a reduced proliferate ability of slp1 deletion mutant in host tissues (Mentlak et al., 2012). In C. higginsianum, LysM effector protein ChELP1 and ChELP2 were required for virulence and ChELP1 RNAi mutants displayed abnormal appressorium in morphology with impaired penetration ability (Takahara et al., 2016). In Verticillium dahliae, LysM effector protein Vd2LysM, but not Vd4LysM, Vd5LysM and Vd6LysM, contributed to virulence on tomato (de Jonge et al., 2013; Kombrink et al., 2017). A LysM protein RsLysM played an important role in Rhizoctonia solani virulence, and heterologous expression of RsLysM in Cercospora beticola increased fungal colonization ability and necrotic lesion size on host (Dolfors et al., 2019). In plant-beneficial fungus Trichoderma atroviride, a seven LysMs effector TAL6 specifically inhibited the germination of Trichoderma spp. and increased the fungus mycoparasitic capacity (Seidl-Seiboth et al., 2013; Romero-Contreras et al., 2019). In Penicillium expansum, PeLysM3 null mutant exhibited slightly lower rate of radial growth, significantly lower percent of germinating spores and shorter germ tubes (Levin et al., 2017). In M. graminicola, LysM effectors regulated pycnidia formation on wheat (Tian et al., 2021). Based on our data, Cg2LysM deletion mutant showed impaired virulence toward rubber tree (Figure 2). Moreover, Cg2LysM deletion resulted in significant reduced conidia production, conidia germination, appressorium formation of C. gloeosporioides, and abnormal primary hyphae which failing to finish the invasion process (Figure 3). These findings demonstrated that LysM effectors could contribute to fungal virulence through regulation of fungal growth and development, including the development of invasion structure.

Chitin is the major component of fungal cell walls and acts as PAMPs which trigger PTI in plant cells (Jones and Dangl, 2006). Most fungal pathogens evaded host immunity through inhibiting chitin-triggered immunity by blocking chitin sensing or signaling (Hu et al., 2021). Chitin-triggered immunity is known to result in induction of ROS burst and pathogenesis-related genes (Mentlak et al., 2012). LysM domain had been demonstrated to have chitin binding activity (Bolton et al., 2008; Jonge et al., 2010). The function of LysM effectors relied on the binding properties of the LysM domain to the fungal chitin to evade host immunity (Kombrink et al., 2011; Marshall et al., 2011). Ecp6 of C. fulvum suppressed chitin-triggered ROS burst in host cells (Jonge et al., 2010). Mg3LysM of M. graminicola inhibited the expression of Pathogenesis-Related (PR) gene and chitinase gene in wheat at the later stage of infection (Marshall et al., 2011). LysM effectors MbLysM5 and MbLysM19 of Marssonina brunnea weakened the expression of PDF1.2 which was chitin-induced in Arabidopsis thaliana (Jiang et al., 2014). SLP1 of M. oryzae prevented the expression of rice Phe ammonia lyase gene, PAL1, and the β-glucanase-encoding gene, rBG (Mentlak et al., 2012). Our group had reported that chitin-triggered immune responses included transcriptional reprogramming of defense-related genes HbPR1 and HbPR5 and ROS accumulation in mesophyll protoplasts of rubber tree (Zhang et al., 2016). In this study, Cg2LysM also showed chitin-binding activity (Figure 5), and the presence of Cg2LysM suppressed chitin-triggered ROS production and the expression of defense-related genes HbPR1 and HbPR5 in rubber tree mesophyll protoplasts (Figures 6, 7). These data confirmed that Cg2LysM, like other LysM effectors, could contribute to invasion through manipulating chitin-triggered plant immunity.

Phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) were known as classical primary defense hormones which played roles in the regulation of basal resistance against multiple pathogens (Bari and Jones, 2009; Robert-Seilaniantz et al., 2011; Pieterse et al., 2012). SA contributed to PTI and ETI, and exogenous SA application induced pathogenesis related (PR) genes expression, endogenous SA levels as well as increased disease resistance (Yang et al., 2015). Phytoalexin Deficient4 (PAD4) was involved in SA accumulation by promoting principal SA biosynthetic enzyme Isochorismate synthase1 (ICS1) gene expression (Cui et al., 2017). 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and 2-oxoglutarate ACC oxidase (ACO) were two key enzymes in ET biosynthesis (Wang et al., 2002). EIN3/EIL1 transcription factors were known as the key regulators of ethylene signaling that sustained a variety of plant responses to ethylene (Dolgikh et al., 2019). Allene oxide synthase (AOS) was a major control point for JA biosynthesis in Arabidopsis thaliana and transcriptional factor Ethylene-Responsive Factor (ERF) was an important component of ET signaling pathway (Laudert and Weiler, 1998; Wasternack and Song, 2017). There are complex interactions among various plant hormone signaling pathways. Despite the complex interaction among SA, JA, and ET defense pathways, the antagonistic interaction between SA and JA, and the synergistic interaction between JA and ET seemed to be dominant (Yang et al., 2015). In this study, the expression profiles of some genes involved in the SA, JA, ET defense response signaling pathway were analyzed in rubber tree leaves inoculated with WT, ∆Cg2LysM and Res-∆Cg2LysM, respectively. The results showed that the genes involved in SA defense signaling (HbPR1, HbPR5, HbNPR1, and HbPAD4) were induced significantly at 24 h in rubber tree leaves post inoculated with ∆Cg2LysM compared to that inoculated with WT and Res-∆Cg2LysM, but the genes involved in both JA and ET defense signaling (HbAOS, HbERF, HbACO, and HbEIN3) were repressed in rubber tree leaves post inoculated with ∆Cg2LysM compared to that inoculated with WT and Res-∆Cg2LysM (Figure 7). These data indicated that Cg2LysM negatively regulated SA mediated defense pathway and positively regulates JA and ET mediated signaling pathway. However, the regulatory mechanism remains unclear.

In fungi, melanin was one of the well-known secondary metabolites which frequently was found in appressorium, a specialized invasion structurer (Bechinger et al., 1999). Melanin played a critical role in pathogenicity through mediating the buildup of high pressure in the appressorium providing the essential driving force for mechanical penetration (Kaverinathan et al., 2017). In our case, we found that the melanin content of ∆Cg2LysM was significantly lower than that of WT (Figures 4A,B). This is the first report on the function of LysM effector in fungal melanin. In Colletotrichum gloeosporioides, a polyketide synthase CgPks1 and a scytalone dehydratase CgSCD1 were involved in melanin synthesis and pathogenicity, and CgPks1 contributed to turgor pressure of the appressorium, but not CgSCD1 (Wang et al., 2020, 2021). Here we detected the transcriptional expression of CgPks1 and CgSCD1 in ∆Cg2LysM, the result showed that level of CgPks1 and CgSCD1 in ∆Cg2LysM was significantly lower than that in WT (Figure 4C), indicating that Cg2LysM modulated melanin synthesis through CgPks1 and CgSCD1. Considering that Cg2LysM is required for successful infection of C. gloeosporioides on onion epidermal cells (Figure 3E), we speculate that the regulation of Cg2LysM on virulence may be related to the development of infection structure and infection process involved in melanin.

In summary, our results demonstrated the function of a LysM effector Cg2LysM on pathogenicity of C. gloeosporioides toward rubber tree through not only self-regulation of fungal conidiation, appressorium formation and melanin mediated infection process, but also manipulation of rubber tree immune responses such as ROS production and transcriptional programs of defense-related genes HbPR1 and HbPR5.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

LZ carried out most of the experiments and analyzed the data. LF cloned the Cg2LysM gene. ZL did the vector construction. QW helped to finish the microscope observation. LZ, BA, and HL wrote the manuscript. CH revised the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by the National Natural Science Foundation of China (Grant Nos. 32060591 and 32260710).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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