Functional and structural characterization of LysM proteins in interactions between fungi and plants

Plants possess an innate immune system that recognizes various types of molecules that accurately betray microbial invasion, also known as invasion patterns (IPs), that include microbe-associated molecular patterns (MAMPs). This recognition occurs through invasion pattern receptors (IPRs) that activate a wide range of immune responses that aim to halt microbial infections. In turn, successful microbes secrete effector proteins to deregulate plant immunity. Chapter 1 introduces the significant role of the major fungal cell wall component, chitin, in the interactions between plants and fungi. On the one hand, this chapter focuses on chitin perception systems that have been characterized in detail in several plant species, while on the other hand the chapter focuses on effector proteins containing lysin motifs (LysM effectors) employed by the tomato leaf mould pathogen Cladosporium fulvum and the wheat Septoria tritici blotch pathogen Zymoseptoria tritici.

To date, all chitin receptors identified in plants belong to either the LysM-containing receptor-like kinases (LysM-RLKs) or LysM-containing receptor-like proteins (LysM-RLPs). For instance, the Arabidopsis LysM-RLK AtLYK5 binds chitin with high affinity and forms a tripartite receptor complex with two further LysM-RLKs, AtLYK4 and AtCERK1, to initiate chitin signaling. Similarly, the rice chitin perception system is composed of the LysM-RLK OsCERK1 in association with the LysM-RLP OsCEBiP. In Chapter 2, by using chitin affinity-purification followed by mass spectrometry we identified two candidate chitin receptor proteins in tomato, the LysM-RLK SlLYK4 and the LysM-RLP SlCEBiP. Silencing of either SlLYK4 or SlCEBiP resulted in significantly impaired chitin responsiveness. Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR-Cas9) we generated mutants of both genes and evaluated their role in chitin signalling. While the function of SlCEBiP needs further assessment because it presently remains unclear whether the mutant that was generated truly disrupts gene function, SlLYK4 was found to play an essential role in mediating chitin signal transduction as SlLYK4 mutants displayed not only greatly compromised chitin-induced immunity but also enhanced susceptibility to C. fulvum infection. We propose that SlLYK4 is a crucial component of the chitin receptor complex of tomato.

To overcome the chitin-induced tomato immunity, C. fulvum secretes the LysM effector Ecp6 to outcompete immune receptors for chitin binding. Two of its three LysMs undergo intracellular LysM dimerization, thus forming a chitin-binding groove (LysM1-LysM3) with ultra-high substrate affinity that goes beyond the affinity of host receptors. The remaining singular LysM domain of Ecp6, LysM2, also displays the capability to bind chitin, albeit with a relatively low affinity that does not permit to outcompete chitin receptors. Chapter 3 aims to investigate whether LysM2 contributes to the virulence function of Ecp6 and how it confers such contribution. Inoculation assays with C. fulvum transformants that express a suite of Ecp6 mutants in the various LysMs revealed that LysM2 contributes to C. fulvum virulence, probably through suppression of chitin-responsive gene expression. Interestingly, a physical interaction of Ecp6 with Arabidopsis AtLYK5 and with tomato SlLYK4 that was characterized in chapter could been demonstrated. Moreover, it appears that while LysM2 confers an interaction with these receptors in a chitin-independent manner, the composite LysM1-LysM3 binding groove contributes to the interaction in a chitin-dependent manner. Thus, besides competing with plant immune receptors for chitin binding, Ecp6 may perturb the assembly of functional chitin receptor complexes that are crucial for the activation of chitin-induced immunity.

Many fungal LysM effectors comprise two LysMs, such as MoSlp1 from the rice blast fungus Magnaporthe oryzae, Vd2LysM from the broad host range vascular wilt fungus Verticillium dahliae, and ChElp1 and ChElp2 from the Brassicaceae anthracnose fungus Colletotrichum higginsianum. They all bind chitin, suppress chitin-triggered host immunity and contribute to fungal virulence. Chapter 4 describes the functional and structural analyses to investigate whether these fungal LysM effectors with two LysMs bind chitin through intramolecular LysM dimerization, like Ecp6, or rather through intermolecular dimerization. As our considerable efforts to obtain a crystal structure of any of these effectors by X-ray crystallography failed since crystal growth did not occur, we hypothesized that these findings could suggest the occurrence of intermolecular chitin binding for these LysM effectors. With DLS measurements and centrifugation assays we were able to confirm that the formation of chitin-induced polymeric complexes for MoSlp1, V2LysM and ChElp2 occurs, potentially mediating the elimination of chitin oligomers at infection sites by precipitation to suppress the activation of chitin-induced plant immunity.

The wheat-specific pathogen Z. tritici encodes three LysM effector proteins, Mg1LysM and Mg1LysM_b that contain a single LysM, and Mg3LysM that possesses three LysMs. Previously, Mg1LysM_b was disregarded as a presumed pseudogene, while Mg1LysM and Mg3LysM were functionally characterized. Chapter 5 provides evidence to show that Mg1LysM_b is not a pseudogene and is functional during wheat colonization. We show that Mg1LysM_b binds chitin, protects fungal hyphae against chitinase hydrolysis and is able to suppress a chitin-induced ROS burst. Fungal inoculation assays reveal that while Mg3LysM confers a major contribution to Z. tritici virulence, also Mg1LysM and Mg1LysM_b contribute to virulence, albeit with smaller contributions, and that all LysM effectors display partial functional redundancy. Thus, we show that Zymoseptoria tritici utilizes three LysM effectors to disarm chitin-triggered wheat immunity.

In Chapter 6, we determined a crystal structure of Z. tritici Mg1LysM to try and explain how this LysM effector protects fungal hyphae against chitinase hydrolysis. Intriguingly, the crystal structure revealed the formation of chitin-independent homodimers as well as chitin-induced dimerization of two Mg1LysM protomers. Based on DLS measurements and centrifugation assays in the presence and absence of chitin oligomers, it could be concluded that Mg1LysM forms a chitin-induced supramolecular structure that, anchored to chitin in the cell wall, may prevent hydrolysis by host chitinases. Interestingly, it could be demonstrated that Mg1LysM_b, as well as RiSLM from the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis that similarly contains a single LysM, polymerize in the presence of chitin as well, suggesting that they also undergo chitin-induced dimerization of ligand independent homodimers.

Besides chitin, several other cell wall polysaccharides have previously been characterized as invasion pattern, such as β-glucan and bacterial peptidoglycan. Chapter 7 synthesizes the findings in this thesis and places them into a broader perspective to highlight the importance of chitin as well as other cell wall components in interactions between plants and microbes.