Ciboria shiraiana, in the filamentous fungal genus Ciboria, family Sclerotiniaceae, order Helotiales of the Ascomycota (Whetzel and Wolf, 1945), is the major fungal pathogen of mulberry sclerotinia diseases, which lead to substantial reductions in production and consequent economic losses (Gray and Richard, 1987; Lv et al., 2011). Under natural conditions at a suitable temperature, the sclerotia break dormancy to generate ascospores that are released and infect the female flowers of mulberry trees. Ascospores germinate on the stigmas of female mulberry trees flowers to form bud tubes, which help ascospores invade into the stigmas and ovaries. Finally, hyphae entangle to form sclerotia in the infected mulberry fruit. The diseased fruit falls into the soil, and the sclerotia stay dormant in the soil until conditions are suitable for the next infection cycle (Lv et al., 2019).

Sclerotia play central roles in the life history of C. shiraiana, and they are necessary structures for the long-term pathogenicity of C. shiraiana. Sclerotia can germinate into fruit body to produce millions of air-borne ascospores, which are vital to the maintenance and spread of diseases in the field (Yu et al., 2012). Owing to the existence of sclerotia, sclerotinia diseases have become serious and hard to control. The compound appressorium is a multicellular infection structure that differentiates from the top of a hypha formed by pathogen. It attaches to the host surface and penetrates the plant cuticle, which helps the pathogen colonize the host plant (Uloth et al., 2016). Appressoria directly affect the pathogenic ability of the pathogen on the host plant. Therefore, it is necessary to study the growth and development of sclerotia and appressoria.

Many transcription factors have been identified as being involved in sclerotia and appressoria development in phytopathogens. For example, GATA-type zinc-finger transcription factor SsNsd1, Forkhead box transcription factor SsFKH1, MADS-Box transcription factor BcMADS1, and Homeobox transcription factor MoHox2 (Kim et al., 2009; Zhang et al., 2016; Fan et al., 2017; Li et al., 2018). The APSES-type transcription factors (including Asm1, Phd1, Sok2, Efg1, and StuA) are fungi-specific transcription factors that play key roles in growth and development, secondary metabolite synthesis, and morphological transitions in fungi (Miller et al., 1992; Gimeno and Fink, 1994; Ward et al., 1995; Aramayo et al., 1996; Stoldt et al., 1997). In the phytopathogen Magnaporthe oryzae, APSES protein MoStu1 interacts with MoSom1 and MoCdtf1. It participates in the cyclic adenosine monophosphate (cAMP)/PKA signaling pathway to regulate hyphal growth and conidial formation, and it reduces pathogenicity by delaying appressoria formation (Nishimura et al., 2009; Yan et al., 2011). Additionally, in M. oryzae, the function of another APSES transcription factor has been reported. Abnormal hyphae are produced in MoSwi6 mutants owing to changes in chitin synthesis and a reduction in melanin. Moreover, the absence of MoSwi6 results in abnormal conidia, the inability of appressoria to pierce and weakened pathogenicity. MoSwi6 interacts with MoMps1 in the mitogen-activated protein kinase (MAPK) pathway and participates in downstream signal transduction (Qi et al., 2012). In addition, the StuA protein is involved in the virulence, spore to hyphal formation process and the regulation of pks1 and lac1 gene expression levels in the melanin-synthesis pathway of Ustilago maydis (Iyer et al., 2002; García-Pedrajas et al., 2010; Baeza-Montañez et al., 2015). The hyphal growth rates of Fusarium graminearum are inhibited in StuA mutants and conidia, which affect pathogenicity, by reducing the production of mycotoxin deoxynivalenol (Lysøe et al., 2011). The StuA protein in Leptosphaeria maculans not only participates in hyphal growth, spore formation, and pathogenicity, but it also affects effector gene expression levels (Soyer et al., 2015). The APSES protein Sok2 is the target of the cAMP/PKA pathway in Ashbya gossypii, and it is also an important positive regulator of sporulation (Wasserstrom et al., 2017). The APSES-like protein Vst1 has been identified in Verticillium dahliae and Verticillium nonalfalfae, and it is involved in the synthesis and pigmentation of melanin and the formation of spores (Sarmiento-Villamil et al., 2018). FvStuA is a regulator of sporulation, toxin synthesis, and virulence in Fusarium verticillioides (Rath et al., 2020).

APSES-type transcription factors play important roles in the growth, development, and virulence of phytopathogens. However, there are still limited studies that functionally characterize of APSES-type transcription factors in C. shiraiana. In this work, we used an RNA interference (RNAi) strategy to characterize the APSES-type transcription factor CsXbp1, which is a key component of the sclerotial development and pathogenicity in C. shiraiana.

Mulberry is widely planted in the Eurasian continent and an important tree used for the rearing of domesticated silkworm (He et al., 2013; Jiao et al., 2020). There are multiple active compounds in its fruits, such as anthocyanins, polysaccharides, and vitamins, which are beneficial to human health (Donno et al., 2015; Chang et al., 2018). The necrotrophic pathogen C. shiraiana is the main threat to mulberry fruits. A variety of methods have been used to control this pathogen, and among them, the biological control has the advantages of safety and efficiency. To develop biological control methods, the underlying mechanisms involving sclerotia and appressoria should be more comprehensively characterized. This could promote the development of some new biological control methods. The sequencing and analyses of the C. shiraiana genome have made it possible to identify potential target genes of biological control (Zhu et al., 2021). Here, we characterized CsXbp1, a fungal-specific APSES-like transcription factor in C. shiraiana. A series of experiments were conducted to explore its functions.

The Saccharomyces cerevisiae Xbp1 protein is a homolog of Swi4 and Mbp1, which are cell-cycle regulators (Mai and Breeden, 1997). In budding yeast, the Xbp1 protein decreases the expression levels of cyclin CLN1-3, CYS3, and SMF2 genes by combining with their promoter regions. Furthermore, the inhibition of CLN1 gene expression promotes sporulation (Mai and Breeden, 2000). Under nutritional deficiency conditions, Xbp1 plays a pivotal role during the transition from the cell cycle state to quiescence (Miles et al., 2013). Five APSES transcription factors (Afp1, Stu1, Mbp1, Swi6, and Xbp1) have been identified in Histoplasma capsulatum. The Xbp1 protein is specifically expressed in the yeast form but not during the mycelial phase (Longo et al., 2018). As far as we know, there is no research on the Xbp1 protein’s functions in plant pathogenic fungi. Here, we identified and functionally characterized an APSES transcription factor CsXbp1. We confirmed that CsXbp1 localized to the nucleus. As the transcriptional regulation activities of cells were mainly concentrated in the nucleus, it was reasonable that the transcription factor CsXbp1 was located in the nucleus. Further demonstrate that CsXbp1 plays an indispensable role in different stages of fungal growth and development and in pathogenicity.

Sclerotia are asexual dormant structures that can survive for several years (Smith et al., 2015). Their exitance is one of the main reasons why pathogenic fungi are difficult to eradicate. Among them, S. sclerotiorum is the most notorious. The number and weight of mature sclerotia formed in the RNAi strains showed obvious downward trends, which indicated that CsXbp1 was involved in the formation and development of sclerotia (Figures 4B,C). Thus, it was speculated that the decreased number of sclerotia would reduce the probability of long-term survival and the release of ascospores. Furthermore, the reduction in the initial infection capability and spreading area may effectively alleviate the plant infection by the pathogen. The APSES-like protein Vst1 is involved in the production of microsclerotia in V. dahliae, and it might be regulated by G-protein/cAMP signaling and MAPK cascades (Sarmiento-Villamil et al., 2018). The expression levels of key genes involved in sclerotia formation were reduced in the RNAi strains (Supplementary Figure S3), indicating that the CsXbp1 may regulate sclerotial formation by participating in the cAMP-signaling pathway (Galagan et al., 2003; Rollins, 2003; Jurick Ii et al., 2004; Erental et al., 2007; Jurick and Rollins, 2007).

The necessity of CsXbp1 for the pathogenicity of C. shiraiana could be observed in the inoculation assays. The pathogenicity of C. shiraiana to host plants was attenuated in the CsXbp1 RNAi strains (Figure 6A). This could, in part, result from the slow growth of the CsXbp1 RNAi strains’ hyphae, but other factors should be inolved. Oxalic acid (OA) is a significant virulence factor of pathogens (Xu et al., 2015). The pathogen secretes a large amount of OA to change the pH value in plants, aid the infection process (Bolton et al., 2006). We conducted bromophenol blue-staining experiments to determine the ability of C. shiraiana to secrete OA and found that there were no significant differences in the OA production between CsXbp1 RNAi strains and the WT strain (Supplementary Figure S5), indicating that the decrease in the pathogenicity of the CsXbp1 RNAi strains were not correlated with the OA level. Additionally, appressoria are the differentiated structures of hyphae that infect hosts to help the pathogen penetrate the first host barrier, the cell wall (Talbot, 2019). In this study, at 72h, the WT strain formed significantly more appressoria than the RNAi strains (Figures 5A,B). We then inoculated wounded leaves and found that the inoculated wounded leaves recovered some of their virulence (Figure 6A). The expression levels of key genes related to appressoria were significantly reduced in the RNAi strains (Supplementary Figure S4; Mitchell and Dean, 1995; Amselem et al., 2011; Li et al., 2012). Thus, the lack of pathogenicity in RNAi strains was mainly due to the appressorial developmental defects.

At present, the main method to prevent mulberry fruit from C. shiraiana infection is spraying antifungal chemicals (Lv et al., 2011). However, owing to the negative effects of antifungal chemical use, such as those affecting the environment and food safety, it is becoming increasingly important to adopt more environmentally friendly biological control methods. Molecular breeding is an important strategy to control diseases caused by this pathogen. A HIGS method was proposed as an effective strategy to improve the situation. In this method, small RNAs are produced by the host plant to target transcripts in pathogens (Koch and Wassenegger, 2021). It has been successfully applied in economically important crops, such as wheat and cotton (Cheng et al., 2015; Xu et al., 2018). We constructed the TRV-HIGS system to verify that the transfer of the CsXbp1 gene fragment into host plants improved the resistance to C. shiraiana (Figure 6C). The data presented here showed that CsXbp1 could serve as a candidate gene for mulberry disease-resistance breeding and fungicide target. In addition, the Xbp1 proteins were highly conserved among C. shiraiana, S. sclerotiorum, and B. cinerea. Thus, it was hypothesized that this fragment played a similar role in these two important pathogens (Dean et al., 2012; Liang and Rollins, 2018), which had broad host ranges and caused severe crop damages. Therefore, CsXbp1 has the potential to be a target gene for controlling broad-spectrum plant diseases.

In conclusion, we used an RNA silencing strategy to explore the function of CsXbp1. Our results revealed sclerotial development and pathogenicity regulated by CsXbp1. Meanwhile, we confirmed that CcXbp1 can be used as a potential target for disease control. Thus, HIGS may become a powerful approach to control sclerotinia diseases. The use of RNAi sequences derived from essential and fungal-specific virulence genes provides an attractive strategy and a huge pool of potential new resistance resources for the breeding of disease-resistant plants. In addition, spraying dsRNA to silence key virulence gene in pathogens might provide another effective method to control plant diseases. There should be more other functional characterization of CsXbp1 and in-depth studies to augment the understanding of the molecular mechanisms of CsXbp1 downstream elements to analyze the molecular pathogenic mechanism of C. shiraiana, which, in turn, could be used to develop novel targets for control mulberry sclerotinia disease in the future.

Publicly available datasets were analyzed in this study. This data can be found here: VNFM00000000.

SZ, PZ, and AZ conceived and designed the study. BC provided help in the subcellular localization assay. SM provided help in TRV-HIGS experiments. RL and XW provided the technical assistance. SZ wrote the manuscript. AZ revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Key R&D Program of China (grant number 2019YFD1000604), China Agriculture Research System (grant number CARS-18-ZJ0201), and the National Science Foundation of Chongqing (grant number cstc2019jcyj-bsh0106). We thank reviewers for their kind suggestions.

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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