A Single Laccase Acts as a Key Component of Environmental Sensing in a Broad Host Range Fungal Pathogen

preLight Authors: Annika Schulz & Shamiul Rasel

Background:

Laccases are enzymes that play a crucial role in mediating the interaction between lignin production and degradation in plants and fungi, which is critical to carbon cycling and maintaining healthy soils. In ascomycetes (fungi that belong to the phylum Ascomycota and generally produce sexual spores in sacs (1, 2)), laccases are typically associated with melanin or pigment deposition on fungal tissue, and most laccase knockout mutants within the phylum exhibit reduced pigmentation. However, the effect of laccase knockouts on fungal development and growth varies widely, possibly due to the expanded repertoire of laccases and functional redundancy. Specific laccase genes have been noted to play a role in virulence on host plants, such as in Colletotrichum gloeosporioides, Colletotrichum orbiculare, and Setosphaeria turcica, where individual laccase gene knockouts resulted in reduced virulence. Because laccases are often associated with pigmentation, the lack of melanin/pigment deposition in the knockout is assumed to be responsible for these observed phenotypes. However, no causal relationship has been established.

Sclerotinia sclerotiorum is a well-known fungal plant pathogen that causes rot/lesions in various plant parts. It can attack a wide range of plant species, including important crops like oilseed rape, soybean, and other vegetables (3), causing serious economic losses globally (4). Plant cell wall-degrading enzymes released by S. sclerotiorum soften, hydrolyze, and degrade plant cells (3, 4). S. sclerotiorum’s oxalic acid disrupts host cell metabolism after destroying the plant cell wall (4). In S. sclerotiorum, previous studies found that knocking out genes involved in melanin biosynthesis did not affect virulence. Nonetheless, little is known about the functions of laccases as virulence factors.

In this preprint, a laccase gene called Sslac2 was identified in S. sclerotiorum and found to be highly upregulated during infection of soybean plants. This study discusses the role of Sslac2 as a global regulator of environmental sensing, affecting every stage of development, infection, and response to environmental cues, which is unlike the laccase gene in a closely related species. The preprint also highlights the potential for targeting fungal laccases to increase resistance in host plants and considers the broader context of laccases within fungal plant pathogen biology.

Key findings

 

Figure 1. Composition of several figures from Westrick et al. (A) Sslac2 is highly upregulated during early stages of infection. (B) Mutants lacking Sslac2 produce malformed appressoria. (C) Mutants lacking Sslac2 are essentially unable to cause disease. (D) WT shows a transcriptional shift in response to GrSt. ΔSslac2 is unresponsive to GrSt and shows a lack of differential gene expression compared to WT. (E) ΔSslac2’s hyphal cell wall is resistant to protoplasting upon treatment with cell wall degrading enzymes. (F) Silencing Sslac2 reduces the size of lesions in infected plants. The reddening around the lesion indicates a resistance response from the host plant.

 

Sslac2 is upregulated during infection and implicated in fungal growth

Fungal laccases have been previously implicated in plant pathogenicity, which lead Westrick and colleagues to investigate the seven laccase-encoding genes in S. sclerotiorum. Comparing the regulation of these genes during infection (when grown in planta vs. in vitro), Sslac2 was the only laccase markedly upregulated (Fig. 1A). This upregulation likely occurs when S. sclerotiorum contacts the plant.

The Sslac2 mutant S. sclerotiorum strain (ΔSslac2) was unable to produce sclerotia (masses of hyphae used for survival (5)) and had significantly decreased laccase activity that was not rescuable with other laccase homologues. These results indicate that when S. sclerotiorum is present on solid surfaces (i.e., plants), it mainly uses Sslac2 for growth and development.

 

ΔSslac2 mutants are essentially non-pathogenic

ΔSslac2 mutants had extremely malformed appressoria (structures formed by the fungus for the purpose of penetrating the host), resulting in difficulty perforating and invading plant tissue. Also, they produced less oxalic acid, which is important for host tissue acidification, cell death inducement, and host defense subversion (Fig. 1B). These attributes, along with additional suspected defects, rendered ΔSslac2 essentially non-pathogenic (Fig. 1C). The authors speculated that ΔSslac2’s virulence defects were likely stemming from other issues, like a hindered ability to respond to environmental stress (induced by the host). In order to analyze this, they compared the gene expression profiles of ΔSslac2 and the WT. When in the presence of GrSt (soybean green stem extract), the WT’s gene expression profile – unlike the mutant’s – showed a major transcriptional shift, representing its response to the proteins, carbohydrates, and chemical signals from the plant (Fig. 1D). Mutants ΔSslac1 and 2 had significantly fewer differentially expressed genes compared to the WT and were essentially non-responsive to GrSt (Fig. 1D).

 

ΔSslac2 mutants cannot properly sense and respond to host-induced environmental stress

Westrick and colleagues next sought to establish the factors contributing to ΔSslac’s environmental sensing defect. Because ascomycete fungi laccases are generally implicated in cellular detoxification and cell wall remodeling, they analyzed the mutant’s sensitivity to different plant antifungal compounds and cell wall stressors. When S. sclerotiorum was treated with toxic plant-derived compounds, the ΔSslac mutants were significantly less resistant than the WT. When treated with cell wall stressors, the ΔSslac mutants were again significantly less resistant. The authors next investigated the basis of ΔSslac2’s increased susceptibility to cell wall stressors. They found that ΔSslac2 was significantly more resistant to protoplasting by cell wall degrading enzymes (Fig. 1E), had structural differences in hyphae/hyphal growth, including decreased hyphal hydrophobicity, and had textural differences in their extracellular matrix (ECM).

Westrick and colleagues observed decreased radial growth for ΔSslac2 grown on PDA (potato dextrose agar) plates but no decrease in mass in liquid media, indicating a specific radial growth defect. They also observed differences in hyphae for ΔSslac2 cultures. ΔSslac2 had increased aerial hyphae that, unlike the WT, grew upwards over the plate dividers and downward into the agar. Because the WT would instead become dormant and begin building survival structures (like sclerotia), the authors hypothesized that Sslac2 may play a role in sensing environmental signals for dormancy or sclerotia production. In order to assess this, they needed to determine whether the differences in ΔSslac2’s radial growth defect and hyphae (agar penetration) were due to issues with directional growth or with responding to environmental dormancy signals. Unlike when grown on PDA, ΔSslac2 grown on cellophane did not have decreased radial growth. This result, taken into account with the lack of sclerotia production, suggests that ΔSslac2 cannot sense the environmental signals for dormancy and instead grows randomly. Therefore, Sslac2 may be implicated in hyphal thigmotropism and differentiation.

 

Host-induced gene silencing of Sslac2 reduces the virulence of S. sclerotiorum

Silencing Sslac2 via VIGS (vector-induced gene silencing) decreased the sizes of the lesions formed on plants by S. sclerotiorum. There was also visible reddening around the lesions on the soybean plants’ stems that were inoculated with a Sslac2 silencing vector, which indicated a host defense response from the plant. These results suggest that silencing Sslac2 reduces the virulence of S. sclerotiorum and allows the host to spend more time mounting a defense (Fig. 1F). Because VIGS was successful, targeting Sslac2 via HIGS (host-induced gene silencing) might be a viable method of disease control.

 

Summary

Overall, this preprint determined that Sslac2, a laccase present in the fungal plant pathogen S. sclerotiorum, is a key player in virulence. It contributes to fungal growth and development and environmental signal sensing, allowing the pathogen to successfully infect its host. Because of these critical roles in virulence and the fact that silencing Sslac2 resulted in decreased virulence, Sslac2 may be an attractive target for fungal disease control in plants.

 

What we like:

This study employs a combination of genetic and biochemical approaches to investigate the function of a particular laccase enzyme. The methods used are well-established in the field of fungal biology, which adds credibility to the study, and the conclusions drawn are well-supported by the results. This study is especially interesting, as little research has been done to establish the roles of secreted fungal laccases in the pathogenesis of plants. The findings indicate a relatively novel role for Sslac2 in environmental sensing that is important for the virulence of S. sclerotiorum. Understanding the mechanisms that underpin fungal pathogens’ virulence is essential for developing effective strategies to control their spread. Determining the roles of Sslac2 in virulence opens the door to disease treatment via targeting Sslac2, which the authors demonstrate may be feasible using HIGS. Considering S. sclerotiorum infects a broad host range of plants, including many crops, treatment has important implications in agriculture and the field of plant pathology.

 

Future directions and questions:

The authors state a few goals/possibilities for future work, including determining substrates of Sslac2 and the specific methods through which Sslac2 functions to regulate thigmotropism and environmental signal response. They also plan on using gene silencing to attempt to target Sslac2 and other laccases to treat fungal infection of plants. Another beneficial future direction could include testing laccase deletion mutants on multiple types of plants grown in various conditions–this would help to identify potential varying roles of laccases when subjected to differing conditions.

We wonder what other proteins or pathways may be involved in environmental sensing and how they interact with the laccase-mediated pathway. We also are interested in the potential implications of deleting Sslac2 (and other laccases) on general cellular processes.

 

References:

(1) M. McConnaughey, (2014). Physical Chemical Properties of Fungi, Reference Module in Biomedical Sciences, Elsevier, ISBN 9780128012383, https://doi.org/10.1016/B978-0-12-801238-3.05231-4.

 

(2) Nicholas P. Money, (2016). Chapter 1 – Fungal Diversity, Editor(s): Sarah C. Watkinson, Lynne Boddy, Nicholas P. Money, The Fungi (Third Edition), Academic Press, Pages 1-36, ISBN 9780123820341, https://doi.org/10.1016/B978-0-12-382034-1.00001-3.

 

(3) Daohong Jiang, Yanping Fu, Li Guoqing, Said A. Ghabrial., (2013). Chapter Eight – Viruses of the Plant Pathogenic Fungus Sclerotinia sclerotiorum. Editor(s): Said A. Ghabrial, Advances in Virus Research, Academic Press, Volume 86, Pages 215-248. https://doi.org/10.1016/B978-0-12-394315-6.00008-8.

 

(4) Yang, C., Li, W., Huang, X., Tang, X., Qin, L., Liu, Y., Xia, Y., Peng, Z., & Xia, S., (2022). SsNEP2 Contributes to the Virulence of Sclerotinia sclerotiorum. Pathogens (Basel, Switzerland), 11(4), 446. https://doi.org/10.3390/pathogens11040446.

 

(5) Money, Nicholas P., (2023). Chapter 2 – Fungal Cell Biology and Development, Editor(s): Sarah C. Watkinson, Lynne Boddy, Nicholas P. Money, The Fungi (Third Edition), Academic Press, Pages 37-66, ISBN 9780123820341, https://doi.org/10.1016/B978-0-12-382034-1.00002-5.

 

Reference for the preprint this preLight covers:

Nathaniel M. Westrick, Eddie G. Dominguez, Christina M. Hull, Damon L. Smith, Mehdi Kabbage. A Single Laccase Acts as a Key Component of Environmental Sensing in a Broad Host Range Fungal Pathogen. bioRxiv 2023.01.12.523834, https://doi.org/10.1101/2023.01.12.523834.

 

 

 

 

 

doi: Pending

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