Recent advances in virulence of a broad host range plant pathogen Sclerotinia sclerotiorum: a mini-review

Sclerotinia sclerotiorum is a typical necrotrophic plant pathogenic fungus, which has a wide host range and can cause a variety of diseases, leading to serious loss of agricultural production around the world. It is difficult to control and completely eliminate the characteristics, chemical control methods is not ideal. Therefore, it is very important to know the pathogenic mechanism of S. sclerotiorum for improving host living environment, relieving agricultural pressure and promoting economic development. In this paper, the life cycle of S. sclerotiorum is introduced to understand the whole process of S. sclerotiorum infection. Through the analysis of the pathogenic mechanism, this paper summarized the reported content, mainly focused on the oxalic acid, cell wall degrading enzyme and effector protein in the process of infection and its mechanism. Besides, recent studies reported virulence-related genes in S. sclerotiorum have been summarized in the paper. According to analysis, those genes were related to the growth and development of the hypha and appressorium, the signaling and regulatory factors of S. sclerotiorum and so on, to further influence the ability to infect the host critically. The application of host-induced gene silencing (HIGS)is considered as a potential effective tool to control various fungi in crops, which provides an important reference for the study of pathogenesis and green control of S. sclerotiorum.


Introduction
Sclerotinia sclerotiorum is a typical necrotrophic nutrient fungus that belongs in taxonomic status to the Fungi, Ascomycota, Discomycetes, Helotiales, Sclerotiniaceae, Sclerotinia.An early study determined that Dictyostelium can infect more than 450 plant species in 75 families, posing a threat primarily to dicotyledonous crops such as sunflowers, soybeans, canola, edible dry beans, chickpeas, peanuts, dry beans, lentils, and a variety of vegetables, but also to monocotyledonous plants such as onions and tulips (Boland and Hall, 1994).Recent research has found that it can grow in rice, wheat, barley, oat, and corn (Tian et al., 2020).
The disease caused by S. sclerotiorum is called Sclerotinia.More than 60 names have been used to refer to diseases caused by this fungal pathogen (Purdy, 1979), including cotton rot, water soft rot, stem rot, abscission, crown rot, flower wilt, and the most common white mold.Zhu et al. 10.3389/fmicb.2024.1424130Frontiers in Microbiology 02 frontiersin.orgS. sclerotiorum is a representative vegetative plant pathogenic fungus with complex pathogenic mechanism.The role and mechanism of oxalic acid, cell wall degrading enzymes, and secretory proteins secreted by S. sclerotiorum in the process of infection have been the focus of reports previously.However, the molecular basis of the pathogenesis of S. sclerotiorum is imprecise and remains a subject of ongoing research.In this paper, the recent advancements in the virulence of S. sclerotiorum have been reviewed, which can serve as an important source of information for molecular research of this organism.

The infection process of Sclerotinia Sclerotiorum
Sclerotinia sclerotiorum (Lib.) de Bary is one of the most dangerous and common plant-killing organisms in the world.This fungus may be found all across temperate, tropical, and dry environment (Lehner et al., 2017).Sclerotia consists of three distinct layers, the thick-walled pigmented cortex, the thin-walled cortex and the white medulla (Clarkson et al., 2004).As its name indicates, the fungus produces sclerotia, which are long-lived melanized resting structures.Sclerotia can germinate in two ways, carpogenically to form apothecia from which ascospores are liberated or myceliogenically to produce hyphae.The fungus is homothallic and no asexual spores are produced (Hegedus and Rimmer, 2005).S. sclerotiorum generally exists in the form of sclerotia, and the existence of ascospores and mycelium form maintains a shorter time (Bolton et al., 2006).When environmental conditions are fit for germination, sclerotia may germinate myceliogenically to produce hyphae that infect the lower parts of plants, or germinate carpogenically to produce apothecia and release ascospores into the air (Behnam et al., 2007;Fernando et al., 2007;Zhang M. et al., 2021).When infected in the form of ascospores, only combined with multiple infection sites, can the successful infection feature be formed.For example, flowers or flower parts provide an appropriate nutritional basis for the initial colonization of ascospore inoculants.The undamaged host surface is infected by hyphae extending from the matrix (Sutton and Deverall, 1983).Once infection has been established, watery lesions with distinct edges may first appear on stems, leaves, petioles, and reproductive organs.These lesions are then followed by wilting, bleaching, and shredding.Typically on the surface of the infected tissue as well as in soft host tissue or lumen, cotton hyphae build up into pea-sized aggregates that eventually develop into hard, black sclerotia (Heffer and Johnson, 2007).When the stem is completely girdled and covered by the whitish mycelial growth all over, the plant wilts and dries.The fungus produces symptoms on all the aerial plant parts but the most destructive one is produced on the stem.Usually, the plants are attacked at flowering stage and once the pathogen is established in the field, it is very difficult to eliminate it due to the stubborn soil-borne sclerotia and wide host range (Bolton et al., 2006).
Strong pathogenicity enable S. sclerotiorum to virtually completely invade all plant tissues with its mycelium.Importantly, the fungus affects hundreds of monocotyledons and dicotyledones and is not restricted to any one host (Jahan et al., 2022).Infection can potentially begin with mycelial development from sclerotia on the soil's surface.After colonizing dead organic debris, the germinated hyphae infect nearby living plants (Hegedus and Rimmer, 2005).Plant pathogen S. sclerotiorum is extremely destructive, and its infection can result in substantial yield loss, the drop in the quantity and quality of affected crops, and other negative economic effects (Fernando et al., 2007).

Pathogenicity factors
Sclerotinia sclerotiorum is a typical necrotrophic plant pathogenic fungus with complex pathogenic mechanism.At present, the main pathogenic factors are classified as plant cell-wall-degrading enzymes(CWDEs), oxalic acid and effector protein secreted by S. sclerotiorum.Firstly, S.sclerotiorum increases its colonization by secreting CWDEs, which plays an important role in the early stage of infection.Oxalic acid, a key pathogenic factor, is secreted in large quantities in the early stage and plays an important role in pathogenicity.In addition, S. Sclerotiorum also secretes some effector proteins to promote infection in its fight against plants.

Oxalic acid, a key factor in the early pathogenesis of Sclerotinia Sclerotiorum
Oxalic acid is considered to be a key factor in the early pathogenesis of many necrotrophic fungi.S. sclerotiorum involves the production and accumulation of oxalic acid at the early stage of plant susceptibility (Figure 1).Godoy et al. (1990) studied the role of oxalic acid in the pathogenicity of S. sclerotiorum in 1990, the oxalic aciddeficient mutants were obtained by UV mutagenesis and lost their pathogenicity.Moreover, the pathogenicity of the mutants was restored after the addition of sodium succinate.
More studies revealed the role of oxalic acid in the pathogenesis of S. sclerotiorum.Oxalic acid can produce reactive oxygen species (ROS) and induce programmed cell death (PCD).In contrast, during the initial stage of S. sclerotiorum infection of host plants, S. sclerotiorum produces a reducing environment in host cells through oxalic acid inhibiting host defense responses, including suppression of ROS bursts and callose deposition, similar to compatible bionutrient pathogens.However, once an infection is established, oxalic acid induces ROS production in the plants, resulting PCD in the host tissue (Williams and Stelfox, 1980).Oxalic acid can regulate the PH of the environment to facilitate the infection of S. sclerotiorum.Sclerotinia sclerotiorum can cause infection and disease at least in some plants as long as the environmental pH is low enough (Bateman and Beer, 1965).Oxalic acid prevents stomatal closure in the host plant.The results showed that when the leaves of Vicia faba were infected with S. sclerotiorum mutants lacking oxalic acid, the stomata were partially closed (Guimarães and Stotz, 2004).Besides, Oxalate prevents stomatal closure caused by abscisic acid (ABA).In Arabidopsis, compared to wild type plants, mutants abi1, abi3, abi4, and aba2 are more vulnerable to oxalate-deficient S. sclerotiorum, indicating that ABA is necessary for Sclerotinia resistance (Guimarães and Stotz, 2004).Oxalic acid may be able to quench calcium ions generated during cell wall collapse to shield developing hyphae from the harmful calcium concentrations in the infection region.Heller and Witt-Geiges (2013) suggest that calcium was transferred to the older parts of the mycelium and detoxified by forming nontoxic, stable oxalate crystals and they proposed an infection model in which oxalic acid plays a detoxifying role in the late stage of infection.

Cell wall degrading enzymes play a vital role in pathogenic fungi invading host
Plant cell wall is an important place for interaction between host and pathogenic fungi, and plays a vital role in the process of plant pathogenic fungi invading host.The pathogenic fungi secrete a series of cell wall degrading enzymes during the process of infecting host plants.They can not only take up nutrition, but also degrade host tissue during the pathogenic process of pathogenic fungi.Sclerotinia sclerotiorum releases hydrolases, which degrade the cuticle, the middle layer, and the primary and secondary cell walls in turn.It was found that the cuticle-stripped leaves infected more quickly, so the cuticle could act as a barrier against Sclerotinia infection.
Sclerotinia sclerotiorum can secrete different cell wall degrading enzymes, especially Polygalacturonases (PGs), when it infects host plants.According to reports, four endo-polygalacturonase (PG) genes (SsPG1, SsPG3, SsPG5 and SsPG6) and two exo-PGs genes (SsXPG1 and SsXPG2) were identified in B. napus during S. Sclerotiorum infection (Li et al., 2004).The factors affecting the regulation of genes encoding polygalacturonase 1(SsPG1) and a newly identified keratinase (SsCUTA) were studied.In vitro, SsCUTA transcripts were detected within 1 h after leaf inoculation, and their expression was mainly controlled by the contact of mycelium with solid surface.The expression level of the keratinase-encoding gene SsCUTA of S. sclerotiorum was significantly up-regulated at 1 h after infection.Expression of SsPG1 was moderately induced by contact with solid surfaces, including leaves, and as infection progresses, expression of SsPG1 was limited to the extended margins of the lesion (Dallal Bashi et al., 2012).Both SsPG3 and SsPG6 induced light-dependent necrosis in Arabidopsis thaliana leaves (Dallal Bashi et al., 2012).

Effector proteins, critical components in the effective pathogenesis of Sclerotinia Sclerotiorum
In the process of antagonism between plant and pathogen, the pathogen will secrete some proteins into plant cells.These proteins are called "Effector proteins, " which can make the pathogen successfully infect the plant.These "Effector proteins, " have been found in a variety of plant pathogenic fungi and exhibit many different functions according to the life style of the fungi.
SsITL encodes a protein containing 302 amino acid residues, which belongs to the integrin-terminal domain superfamily.The expression of SsITL gene increased sharply during the early stage of infection and after the gene silencing, the pathogenicity of S. sclerotiorum decreased.The host plants overexpressing SsITL gene were more susceptible.Further studies showed that SsITL protein was involved in the inhibition of JA/ET signaling pathway-mediated local and systemic disease resistance in S. sclerotiorum.Therefore, SsITL played a similar role as an effector in S. Sclerotiorum pathogenesis (Wang et al., 2009).Two genes, SsNEP1 and SsNEP2, encoding necrotic and ethylene-induced polypeptides (NEPs), were identified in S. sclerotiorum.During infection, the expression of SsNEP1 was increased.The expression of SsNEP2 was induced by contact with solid surfaces and occurs in necrotic areas and the leading edge of infection.Expression of SsNEP2 was dependent on calcium and cyclic AMP signaling, instead, compounds that affected these pathways reduced or eliminated SsNEP1 expression with partial or complete loss of virulence (Dallal Bashi et al., 2010).A small cysteine-rich protein, SsCVNH, has been shown to be crucial for virulence and sclerotium development (Lyu et al., 2015a).Another small cysteine-rich protein A model of oxalic acid (OA)-induced infection.S. sclerotiorum secretes large amounts of oxalic acid during infection.In the early stage of infection, oxalic acid blocks the closure of the plant stomata to promote infection, and then breaks down the plant barrier, at which point the host secretes ABA to shut down the plant stomata to resist infection.Oxalic acid can inhibit plant autophagy, through the outbreak of producing reactive oxygen species (ROS) in the later stage to inhibit the host defense response, and finally cause the programmed cell death (PCD) of plants.In addition, oxalic acid can preserve ca 2+ generated by cell wall collapsing in the form of calcium oxalate in the vacuole, playing a detoxification role.Zhu et al. 10.3389/fmicb.2024.1424130Frontiers in Microbiology 04 frontiersin.orgSsSSVP1 in S. sclerotiorum has been identified as a secreted protein and its targeted silencing resulted in reduced virulence (Lyu et al., 2016).

Reported genes regulating virulence in Sclerotinia Sclerotiorum
The virulence of S. Sclerotiorum is related to the growth and development of its appressorium and its ability to infect plants.Studies on the virulence of S. sclerotiorum to date have been shown as follows (Table 1).In the regulation of pathogenicity in S. sclerotiorum, several genes play pivotal roles.SsTrx1 contributes to enhancing the pathogenicity of the fungus and its tolerance to oxidative stress (Rana et al., 2021), while overexpression of SsYCP1 promotes S. sclerotiorum infection and enhances its pathogenicity (Fan et al., 2021a).SsNACα regulates the expression of polygalacturonase, thus impacting the pathogenicity of S. sclerotiorum (Li et al., 2015).Additionally, SsERP1 modulates the virulence of S. sclerotiorum by regulating the ethylene pathway (Fan et al., 2021b).Moreover, SsSte12 influences fungal hyphal growth, adhesion structure formation, and virulence regulation (Xu et al., 2018).SsPDE2 plays a regulatory role in oxalate accumulation, adhesion structure formation, and virulence (Xu et al., 2023).Furthermore, SsXyl1, encoding endo-β-1,4-xylanase, plays a crucial role in sclerotium formation and infection processes (Yu et al., 2016).SsNsd1 regulates the morphology of S. sclerotiorum hyphae, appressorium structures, sclerotium, and pathogenicity (Li et al., 2018).
The signaling and regulatory factors of S. sclerotiorum involve the regulation of multiple genes.SsTOR is responsible for controlling cell wall integrity and virulence, and it participates in the rapamycin target signaling pathway (Jiao et al., 2023).SsCat2 regulates catalase activity, affecting aspects such as cell membrane dryness and pathogenicity (Huang et al., 2021).SsPKA2 and SsPKAR are involved in cAMP signaling, playing roles in growth and virulence (Yu and Rollins, 2022).Moreover, SsAMS2 contains a GATA-box domain, regulating hyphal growth, adhesion structure formation, and virulence (Liu et al., 2018).Lastly, SsSm1 participates in the MAPK signaling pathway, influencing aspects such as hyphal growth and pathogenicity (Pan et al., 2018).
These genes play critical roles in the metabolic regulation of S. sclerotiorum.Ssoah1 regulates oxalic acid content, affecting the toxicity of S. sclerotiorum (Liang et al., 2015a;Rana et al., 2022).Next, SsSOD1 is responsible for regulating the clearance capacity of ROS, directly impacting the pathogenicity of S. sclerotiorum.Additionally, SsODC1 and SsODC2 are involved in oxalic acid metabolism regulation, playing crucial regulatory roles in forming infection cushions and virulence during the infection process (Xu and Chen, 2013).
In the regulation of cellular structure and function in S. sclerotiorum, SsAGM1 participates in chitin synthesis, influencing cell wall structure and sclerotia formation (Zhang et al., 2022).Subsequently, SsGSR1 encodes glutathione sulfurtransferase, regulating cell wall integrity and the pathogenicity of S. sclerotiorum (Hu et al., 2023).SsCP1 encodes the Cerato-platanin protein, affecting cell death and invasion ability during infection (Yang et al., 2018).Furthermore, as a small secreted protein, SsSSVP1 influences plant cell death and the infection of S. sclerotiorum (Lyu et al., 2016).
In the adaptation to environmental changes and stress responses in S. sclerotiorum, SsNE2, as a novel necrosis-inducing protein, influences the fungal growth, pathogenicity, and ability to respond to environmental stress (Seifbarghi et al., 2020).As we know, thioredoxin reductases play crucial roles in maintaining cellular redox homeostasis.SsTrr1, encoding thioredoxin reductase, participates in oxidative stress responses, thereby affecting the pathogenicity of S. sclerotiorum (Zhang et al., 2019).Additionally, SsEmp24 and SsErv25, as early secretory pathway-related proteins, regulate the protein secretion process, playing important roles in the pathogenicity of S. sclerotiorum.Their absence leads to abnormal fungal growth, sclerotium formation, formation of appressorium, and lower virulence in host plants (Xie et al., 2021).

Host-induced gene silencing of virulence-related genes of Sclerotinia Sclerotiorum
Most eukaryotic organisms possess the RNA interference (RNAi) pathway.In this process, double-stranded RNA (dsRNA) can be processed into small interfering RNA (siRNA), which can induce gene silencing.As RNA can be transferred from plant hosts to related eukaryotic pathogens, the RNAi pathway has been utilized for HIGS to control pathogens.In this scenario, hosts are engineered to express dsRNAs targeting crucial pathogen genes, thus disrupting their successful lifecycle.It is considered to be a potentially effective tool for controlling various fungi in crops (Chen et al., 2016;Zhu et al., 2017;Qi et al., 2018).
With the discovery of more and more virulence-related genes of S. sclerotiorum, many genes can be used to control S. sclerotiorum through HIGS.In tobacco, the authors tested whether the MAPK cascade consisting of SsSte50-SsSte11-SsSte7-Smk1 could serve as a target for disease control in HIGS.SsSte50 is used to make hairpin RNAi constructs.Compared with no-load, the lesion area was significantly reduced.The data showed that the HIGS of SsSte50 was heritable and could be used as a good target for controlling S. sclerotiorum (Tian et al., 2024).Besides, a gene was found to encode a cAMP phosphodiesterase (SsPDE2).The author introduced the construction of HIGS targeting SsPDE2 into N.benthamiana, observing a significant reduction in virulence against S. sclerotiorum.In summary, SsPDE2 may serve as a HIGS target to control stem rot in the field (Xu et al., 2023).Transient silencing of Sscnd1 gene by HIGS mediated by tobacco rattle virus (TRV) can significantly reduce the occurrence of tobacco diseases.Three transgenic Arabidopsis lines with HIGS gene showed a high level of resistance to S. sclerotiorum and reduced the expression of Sscnd1 (Ding et al., 2021).Similarly, a putative protein kinase SsCak1, silencing by TRV-HIGS can reduce virulence and enhance host resistance to S. sclerotiorum (Qin et al., 2023).In rapeseed, three disease-causing genes, the endopolygalacturonase gene (SsPG1), cellobiohydrolase gene (SsCBH), and oxaloacetate acetylhydrolase gene (SsOAH1), were selected as the targets of HIGS, which significantly reduced the transcript levels of the target genes (Wu et al., 2022).A RAS-GTPase activating protein SsGAP1, which plays an important role in sclerotia formation, showed complex appressorium production and virulence.The author observed reduced virulence when they introduced HIGS constructs targeting SsGAP1, SsRAS1, and SsRAS2 in Arabidopsis, as well as in tobacco (Xu 10.3389/fmicb.2024.1424130Frontiers in Microbiology 05 frontiersin.org, 2024).Likewise, S. sclerotiorum thioredoxin 1 gene (SsTrx1) has also been identified as a potential HIGS target through the disease resistance analysis in above two plants (Rana et al., 2021).

Conclusion
The extremely dangerous soil-borne pathogen S. sclerotiorum poses a serious risk to crops used in agricultural production.S. sclerotiorum can not be eradicated in the field due to the lack of resistant breeding and the resistance of S. sclerotiorum strains to fungicides.On the other hand, the negative effects of pesticide residues and environmental pollution are also readily apparent, as they can result in the emergence of drugresistant strains in the field and the death of beneficial organisms.Thus, it will be helpful to regulate S. sclerotiorum to comprehend the pathophysiology of the organism as well as to create and employ diseaseresistant genes in plants.For instance, oxalic acid is a pathogenic component of S. sclerotiorum.Hence, lowering the pathogenicity of S. sclerotiorum can be achieved by reducing oxalic acid production.Moreover, targeting silencing of some proteins, such as Ss-Rhs1, can lead to aberrant colony formation and decreased pathogenicity to the host plant, hence improving host plant resistance to S. sclerotiorum.However, for all control methods, a successful strategy should be based on an in-depth understanding of the pathogenesis of S.sclerotiorum.The application of HIGS targets of known virulence-related genes can effectively provide host resistance to S. sclerotiorum.This may be an effective way to control sclerotinia in the future, providing a new idea for the green control of this broad host range plant pathogen.Plant Pathol. 11, 43-53. doi: 10.1111/j.1364-3703.2009.00571.x Dallal Bashi, Z., Rimmer, S. R., Khachatourians, G. G., and Hegedus, D. D. (2012).Factors governing the regulation of Sclerotinia sclerotiorum cutinase a and polygalacturonase 1 during different stages of infection.Can.J. Microbiol. 58, 605-616. doi: 10.1139/w2012-031 Ding, Y., Chen, Y., Wu, Z., Yang, N., Rana, K., Meng, X., et al. (2022).SsCox17, a copper chaperone, is required for pathogenic process and oxidative stress tolerance of Sclerotinia sclerotiorum.Plant Sci. 322:111345. doi: 10.1016/j. plantsci.2022.111345 Ding, Y., Chen, Y., Yan, B., Liao, H., Dong, M., Meng, X., et al. (2021).Hostinduced gene silencing of a multifunction gene Sscnd1 enhances plant resistance against Sclerotinia sclerotiorum.Front.Microbiol. 12:693334. doi: 10.3389/ fmicb.2021.693334 Erental, A., Harel, A., andYarden, O. (2007).Type 2A phosphoprotein phosphatase is required for asexual development and pathogenesis of Sclerotinia sclerotiorum.Mol.Plant-Microbe Interact. 20, 944-954. doi: 10.1094/MPMI-20-8-0944 FIGURE 1

TABLE 1
Virulence-related genes of S. sclerotiorum.