By Fiona Zhang
The love-hate ‘tug of war’ between plants and fungi has long been one of nature’s most fascinating enigmas. On the one hand, the mutualistic mycorrhizal symbiosis allowed embryophytes to colonise the terrestrial biosphere ca. 500 Mya by expanding the nutrient uptake repertoire of plants;1 on the other hand, pathogenic fungi account for approximately 80% of plant diseases.2 The host-specific, plant-pathogenic, hemibiotrophic filamentous ascomycete fungus: Zymoseptoria tritici, also known as Septoria tritici and formerly as Mycosphaerella graminicola, is the causative agent of the foliar disease of wheat: Septoria tritici Blotch (STB). Each year, STB is estimated to reduce 5-10% of wheat harvest, summed up to a loss of more than 1000 million Euros in the countries UK, Germany, and France alone.3 Due to the magnitude of its economic impact, extensive research has been conducted by plant pathologists to understand the mechanism of action and key effector proteins of Z. tritici. Recently, scientists have identified a key effector protein that supports the pathogenic lifestyle of Z. tritici: Zt3LysM.
Overview of infection cycle
For plant-pathogenic fungi to successfully establish diseases, they need to colonise host tissues by extending their hyphae across host cells, following penetration into their hosts’ tissues 4. Following colonisation and soon after the onset of necrotic symptoms (which include leaf spots, blight, scab, and rots), the final stage of the infection cycle of Z. tritici is the formation of fruiting bodies (pycnidia), which contain spores that can then be dispersed for reproduction.5
There is a time lag between the entry of Z. tritici via the stomata opening into its host (the hexaploid common wheat) and the display of necrotic symptoms.4 This is the initial asymptomatic latent phase, also known as the biotrophic phase, in which wheat leaves appear healthy, apoplastic nutrient composition remains constant, and fungal biomass barely changes.4 During this phase, apoptotic programmed cell death, a crucial component in the immune response of plants, is actively suppressed 6, 7, allowing invasive fungal hyphae to spread throughout substomatal cavities and intercellular spaces of the plant host. Z. tritici then enter host cells and exploit host nutrients to produce pre-pycnidia (precursors of fruiting bodies), which is then followed by necrosis.4, 7 At this point, the dying wheat cells have little to no genetic control over their fate and is destined for death soon after.
Role of Zt3LysM in infection
While previously disregarded as a pseudogene, recent studies identified Zt3LysM (previously named Mg3LysM in literature), as the gene encoding key effector protein that enables Z. tritici to cause disease in Triticum aestivum (the common wheat). Just as the latent phase is crucial for Z. tritici to invade wheat tissues, Zt3LysM is vital for the establishment of this latent phase in the first place.4, 5
The LysM of this effector protein confers its function. When found in plant proteins, LysM mediate pathogen-associated molecular patterns (PAMP)-triggered immunity. Examples of such LysM plant proteins include CERK1 and CEBiP, which are well-conserved and widespread among plant species belonging to distantly related taxa, from Arabidopsis to T. aestivum. They function as PRR (pattern recognition receptors) for chitin (a homopolymer of N-acetylglucosamine that is a major structural component of fungal cell walls 5) to induce plant immunity upon stimulation.9, 10 The signalling cascade they set off ultimately triggers plants to both produce hydrolytic enzymes such as chitinases, which inhibit fungal growth by disrupting its cell wall integrity, and release chitin molecules that act as PAMP to be recognised by other PRRs to set off further immunological response cascades against fungal invasion, including the induction of programmed cell death.5
In contrast, fungal LysM effectors, including the chitin-binding Zt3LysM, are broadly conserved effectors that allow Z. tritici to mask its own chitin to evade detection by wheats. They function as inhibitors of chitin-induced plant immunity in wheat by blocking CERK1 and CEBiP, and therefore any subsequent signalling.11 Without the risk of disruption or destruction immediately after entering its host, Z. tritici can spread throughout extracellular spaces of wheat leaves during the latent phase, and, in 5-10 days, begin to consume and depend on host nutrients for its pycnidia.
The major wheat proteins that Zt3LysM acts on was pinned down by experiments such as that conducted by Lee et al. (2014) using single-gene deletion mutants –∆Zt3– lacking Zt3LysM, to test their virulence on both wild-type (WT) wheats and wheats with deletion of the CEBiP and CERK1 genes.9 While WT wheats were unaffected by ∆Zt3 strains, the deletion of CEBiP and CERK1 genes in wheats somewhat restored the ∆Zt3 strains’ ability to cause necrosis in wheat leaves, despite absence of Zt3LysM.9 This not only sheds light on the role of proteins CERK1 and CEBiP in the immunity of wheat against fungal pathogens, but also shows that Zt3LysM acts on the two LysM wheat proteins: CERK1 and CEBiP, as the deletion of them helps ∆Zt3 strains to regain virulence.9 However, the rate of necrosis development in mutant wheats infected by mutant Z. tritici is less that in WT wheat infected by WT Z. tritici, suggesting that Zt3LysM also interacts with wheat immune proteins other than CERK1 and CEBiP, the identities of which have yet to be discovered. 9, 10
As Zt3LysM is one of the three LysM (lysin motif, an approximately 50-amino acid globular domain8) effector homologues in the Z. tritici genome, researchers were eager to distinguish its role from that of its two homologues –Mg1LysM and MgxLysM– during pathogenesis. Tian et al. (2021) investigated this using Z. tritici single-gene deletion mutants: ∆Zt3, ∆Mg1, and ∆Mgx1 and observing the severeness of necrotic symptoms they induce in wheat hosts.5 Compared to its homologues, Zt3LysM contributes significantly more to the virulence of Z. tritici.5, 9, 12 While both the mutant strains: ∆Mg1 and ∆Mgx1, as well as the double-gene mutant strains: ∆Mg1-∆Mgx1, caused similar levels of necrosis as the WT strains (no apparent decrease in virulence), the ∆Zt3-∆Mg1, ∆Zt3-∆Mgx1, and ∆Zt3 strains were severely compromised in virulence. Without the Zt3LysM gene, wheats were close to unaffected and barely had any visible morphological symptoms of STB.5 Such results suggest that the LysM effectors Mg1LysM and Mgx1LysM are dispensable for the virulence of Z. tritici.5, 9 However, the necrotic symptoms caused by inoculation with the triple-gene deletion mutant ∆Mg1-∆Mgx1-∆Zt3 strains were significantly reduced when compared to those caused by the single-gene deletion mutant ∆Zt3 strains. Combined, such results indicate that while Zt3LysM is the most important LysM effector of Z. tritici in terms of its disease development, Mgx1LysM and Mg1LysM contribute to disease development through redundant functionality: one can substitute the other in function, as they contribute equally to necrosis.5
In addition, Zt3LysM works in conjunction with MgxLysM to produce pycnidia. Tian et al. (2021) determined the role of the LysM effectors in pycnidia formation by measuring the percentage of leaf surface pycnidia coverage in deletion mutants.5 Confoundingly, results revealed that ∆Mg1 strains developed significantly more pycnidia than the WT strains, whereas both the ∆Mgx1 and ∆Zt3 strains produced little to no pycnidia. Furthermore, whereas the double-gene deletion ∆Mg1-∆Mgx1 strains developed an intermediate number of pycnidia, all mutants with ∆Zt3 were devoid of pycnidia. Such data suggest that symptom development (measured by necrosis) does not correlate with fungal colonisation levels (measured by pycnidia coverage) and that the three LysM effectors display differential roles in fungal colonisation. The details of how the three homologue effectors interact to explain the results is yet again another mystery for now. Despite this, Zt3LysM is demonstrated to be important in both the display of necrotic symptoms in the wheat host, and the colonisation levels and is therefore key in the pathogenic lifestyle of Z. tritici.
Is the function of Zt3LysM in Z. tritici specific to pathogenic lifestyles?
While hyphal growth is a prerequisite for the pathogenic lifestyles of plant-pathogenic fungi, many fungal species are pleomorphic (have various vegetative growth forms). The growth forms of Z. tritici includes multi-cellular polar hyphal tip growth, single-celled yeast growth, and propagation by both sexual ascospores and asexual pycnidiospores, which seems unique, but is not atypical of plant-pathogenic fungi.4 The growth forms fungi employ at a particular time is dependent upon various external factors, and controlled switches in growth forms (such as dimorphic switching: between multicellular hyphal and unicellular yeast growth) can be triggered by nutrient availability, temperature, and other factors.4
While the deletion of Zt3LysM severely impedes the ability of Z. tritici to infect wheat, yeast-like growth and other growth forms are unaffected.4, 5, 12, 13 Through deleting the gene encoding the glycosyltransferase, ZtGT2 (a trans-acting sequence downregulating the expression of Zt3LysM) and investigating its effects on both the concentration of effector Zt3LysM and the morphological differences between mutant and WT strains, King et al. (2017) reported that during yeast growth and spore growth phases, Zt3LysM expression levels and Z. tritici morphologies are not affected. Conversely, Zt3LysM was significantly overexpressed during the hyphal growth phase of Z. tritici, suggesting that expression of Zt3LysM is only activated required only during pathogenesis.13 It is hypothesized that, since abnormal cell wall structures were observed in ZtGT2 deletion mutants, that such cell wall differences may mimic inoculation onto plants, leading to the overexpression of Zt3LysM.13 However, there is no evidence supporting this claim due to lack of relevant research.
Zt3LysM homologues and orthologues
Both evolutionary homologues and functional orthologues of Zt3LysM are found in other plant-pathogenic fungi, with the same chitin-binding properties. In fact, the recent discovery of the role of Zt3LysM was due to its homology to known virulence genes in other plant pathogen.10, 13
A well-studied example of a functional orthologue is the effector molecule Avr4 in the tomato leaf mould fungus Cladosporium fulvum.5, 14 The host of Cladosporium fulvum, tomato, also possesses CERK1 and CEBiP genes, further supporting the necessity of LysM effectors in evading detection by CERK1 and CEBiP receptors.9, 12 From an evolutionary perspective, plant LysM receptors and fungal LysM effectors likely developed in the arms race between plant hosts and fungi pathogens.
Examples of Zt3LysM homologue effectors in plant-pathogenic fungi include SnTox1 in another wheat pathogen Parastagonospora nodorum 15, Slp1 in the rice blast fungus Magnaporthe oryzae 16, LysM2 in Clonostachys rosea 17, and many more.5 These all contain LysM, and are variations corresponding to the PRR in their plant host(s).5
The presence of well-conserved homologues and functional orthologues further highlight both the cruciality of LysM effectors in the success of plant-pathogenic fungi, and that using LysM effectors to suppress host immunity is not specific to Z. tritici. While this breakthrough seems like a promising starting point for creating broad-spectrum fungicides targeting LysM effectors, it has limitations. For example, it remains unclear why some filamentous fungi, such as the basidiomycete plant-pathogenic basidiomycete Puccinia graminis (stem rust), lack both genetic homologues and functional orthologues of Zt3LysM, despite being able to extend hyphae across solid surfaces and in plants 13. Such exceptions point to the direction of subsequent research, which will be driven both by the potential to mitigate enormous crop yield losses and the passions of researchers working in this field.
To this day, surprisingly little is known about the cell and molecular biology of Z. tritci. Zt3LysM remains the only effector identified in Z. tritici that is required for its virulence.10 The discovery of the widespread LysM homologues and functional orthologues is an encouraging start, but much remains to be done to understanding the host-pathogen interactions and molecular mechanisms facilitating pathogenesis.
- Morris, J., Puttick, M., Clark, J., Edwards, D., Kenrick, P., Pressel, S., Wellman, C., Yang, Z., Schneider, H. and Donoghue, P., 2018. The timescale of early land plant evolution. Proceedings of the National Academy of Sciences, 115(10).
- Peng, Y., Li, S., Yan, J., Tang, Y., Cheng, J., Gao, A., Yao, X., Ruan, J. and Xu, B., 2021. Research Progress on Phytopathogenic Fungi and Their Role as Biocontrol Agents. Frontiers in Microbiology, 12.
- Fones, H. and Gurr, S., 2015. The impact of Septoria tritici Blotch disease on wheat: An EU perspective. Fungal Genetics and Biology, 79, pp.3-7.
- Steinberg G. Cell biology of Zymoseptoria tritici : Pathogen cell organization and wheat infection. Fungal Genetics and Biology. 2015;79:17-23.
- Tian H, MacKenzie C, Rodriguez‐Moreno L, Berg G, Chen H, Rudd J et al. Three LysM effectors of Zymoseptoria tritici collectively disarm chitin‐triggered plant immunity. Molecular Plant Pathology. 2021;.
- Koeck M, Hardham A, Dodds P. The role of effectors of biotrophic and hemibiotrophic fungi in infection. Cellular Microbiology. 2011;13(12):1849-1857.
- Dickman M, de Figueiredo P. Death Be Not Proud—Cell Death Control in Plant Fungal Interactions. PLoS Pathogens. 2013;9(9):e1003542.
- Buist G, Steen A, Kok J, Kuipers O. LysM, a widely distributed protein motif for binding to (peptido)glycans. Molecular Microbiology. 2008;68(4):838-847.
- Lee W, Rudd J, Hammond-Kosack K, Kanyuka K. Mycosphaerella graminicola LysM Effector-Mediated Stealth Pathogenesis Subverts Recognition Through Both CERK1 and CEBiP Homologues in Wheat. Molecular Plant-Microbe Interactions®. 2014;27(3):236-243.
- McDonald M, McDonald B, Solomon P. Recent advances in the Zymoseptoria tritici—wheat interaction: insights from pathogenomics. Frontiers in Plant Science. 2015;6.
- Hu S, Li J, Dhar N, Li J, Chen J, Jian W et al. Lysin Motif (LysM) Proteins: Interlinking Manipulation of Plant Immunity and Fungi. International Journal of Molecular Sciences. 2021;22(6):3114.
- Marshall R, Kombrink A, Motteram J, Loza-Reyes E, Lucas J, Hammond-Kosack K et al. Analysis of Two in Planta Expressed LysM Effector Homologs from the Fungus Mycosphaerella graminicola Reveals Novel Functional Properties and Varying Contributions to Virulence on Wheat. Plant Physiology. 2011;156(2):756-769.
- King R, Urban M, Lauder R, Hawkins N, Evans M, Plummer A et al. A conserved fungal glycosyltransferase facilitates pathogenesis of plants by enabling hyphal growth on solid surfaces. PLOS Pathogens. 2017;13(10):e1006672.
- de Jonge R, Peter van Esse H, Kombrink A, Shinya T, Desaki Y, Bours R et al. Conserved Fungal LysM Effector Ecp6 Prevents Chitin-Triggered Immunity in Plants. Science. 2010;329(5994):953-955.
- Liu Z, Gao Y, Kim Y, Faris J, Shelver W, Wit P et al. SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual‐function protein that facilitates infection while protecting from wheat‐produced chitinases. New Phytologist. 2016;211(3):1052-1064.
- Mentlak T, Kombrink A, Shinya T, Ryder L, Otomo I, Saitoh H et al. Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease. The Plant Cell. 2012;24(1):322-335.
- Dubey M, Vélëz H, Broberg M, Jensen D, Karlsson M. LysM Proteins Regulate Fungal Development and Contribute to Hyphal Protection and Biocontrol Traits in Clonostachys rosea. Frontiers in Microbiology. 2020;11.