Morels (Morchella spp.), members of the Ascomycetes, Pezizomycetes, Pezizales, Morchellaceae, and Morchella, are commercially important edible mushrooms that are consumed throughout the world (Du et al., 2012). After more than 100years of trials, the commercial cultivation of Morchella in the field was successful (Ower et al., 1986; Dahlstrom et al., 2000; Masaphy, 2010). Since 2012, the industry that cultivates Morchella in China, with Sichuan as a typical example, developed rapidly (Peng et al., 2016). The cultivation area of morels in China has expanded from 200ha in 2012 to 10,000ha in 2020 (Zhao, 2021). The considerable economic benefits of morel cultivation have attracted many farmers and have become their main source of income. The Morchella species currently cultivated in China include M. importuna, M. sextelata, and M. septimelata (Liu et al., 2018). Among them, M. sextelata comprises >90% of the total area of Morchella cultivation (Deng et al., 2021). However, the white mold disease (WMD) caused by Paecilomyces penicillatus (Ascomycetes) seriously harms Morchella (He et al., 2017). WMD outbreak spreads quickly, infecting a large area. In severe cases, 60–80% of Morchella are infected in China, resulting in a decline in their production and commodity value (Chen et al., 2017; He et al., 2017). In addition, a high proportion of P. penicillatus in soil microbial community could inhibit the formation of f Morchella fruiting bodies (Tan et al., 2021a). A large number of farmers suffer financial hardship owing to WMD. Therefore, it is extremely urgent to prevent and control WMD.

Edible mushrooms are primarily cultivated and managed indoors. Therefore, the prevention and control of edible mushroom diseases rely on environmental controls during cultivation, including the control of temperature, light, water, and air, as well as the use of physical and chemical sterilization methods (Gea et al., 2021). Morchella is artificially cultivated in the field (Liu et al., 2018). Hence, the common strategies to prevent and control diseases of edible mushrooms are not effective for the control of Morchella diseases, such as WMD. Similar to other major crops, breeding resistant varieties are an effective way to control mushroom diseases (Kage et al., 2016). Several studies have addressed the immune response of mushrooms to mycoparasites, although this area of research is in its infancy. The activity of laccase, particularly encoded by lcc2 gene, was found to contribute to the metabolism of toxin production in Trichoderma aggressivum and enhanced the resistance to green mold disease in Agaricus bisporus (Sjaarda et al., 2015). A total of 17 simple sequence repeat markers associated with resistance to Mycogone perniciosa in A. bisporus have been established (Fu et al., 2016). A quantitative trait loci analysis was used to identify the locations, numbers, and effects of genomic regions associated with resistance to Lecanicillium fungicola in A. bisporus, and four traits related to resistance were analyzed (Foulongne-Oriol et al., 2012). Further research and data mining will help design classical breeding and genetic modification schemes, thus, producing resistant varieties.

Recently, the genome-wide profiles of several species of Morchella, including M. importuna, M. sextelata, and M. septimelata, as well as that of the pathogen P. penicillatus, were reported (Wingfield et al., 2018; Mei et al., 2019; Tan et al., 2019; Wang et al., 2020). P. penicillatus was found to harbor many CAZymes, particularly clusters of chitinase genes, which could be related to its pathogenicity (Wang et al., 2020). Cheng et al. (2021) reported the regulatory mechanism of interaction between P. penicillatus and M. importuna. However, the lack of a control group of M. importuna at the same developmental stage complicated the conclusions from that study. To date, the genetic and pathogenic mechanisms that underlie the interaction between P. penicillatus and Morchella remain unclear, which makes it difficult to efficiently breed resistant varieties and develop specific biological or chemical methods to control WMD in Morchella. In this study, M. sextelata, the most prevalent species of Morchella in China, was selected as the research object and artificially inoculated with P. penicillatus to develop WMD in the field. The transcriptional changes of P. penicillatus in different infection stages were analyzed. The differences in transcriptional changes were compared between different growth stages of infected and healthy M. sextelata. We aimed to profile the infection mechanism of P. penicillatus, the response mechanism of M. sextelata to P. penicillatus infection, and the candidate defense genes of M. sextelata that protect against P. penicillatus. This will provide theoretical support for the effective control of WMD and reduce the economic risk caused by WMD in the cultivation of Morchella.

Owing to the lack of resistant varieties and the prevalence of P. penicillatus in many regions, WMD poses a serious threat to the cultivation of morels in China. This study identified a number of genes involved in the regulation of the pathogenicity of P. penicillatus to M. sextelata and the response of M. sextelata to infection with P. penicillatus. We defined how P. penicillatus successfully parasitizes M. sextelata and how M. sextelata encodes its immune response to invasion by P. penicillatus.

Mycoparasitic fungi usually produce various fungal CWDEs to lyse the host cell wall, including glucanases, chitinase, and proteases (Gruber and Seidl-Seiboth, 2012). A previous study found that P. penicillatus encodes approximately 300 CAZymes, which could be involved in the degradation of fungal cell walls (Wang et al., 2020). P. penicillatus activated six β-glucanases, three α-mannosidases, and two α-1,6-mannanases when it infected M. sextelata (Figure 3B), indicating that glucanases and mannanases were the primary CAZymes of P. penicillatus that are involved in the degradation of M. sextelata cell walls. Only two chitinases (GH18) were identified in P. penicillatus, even though they were the most annotated CAZyme family in the P. penicillatus genome (Wang et al., 2020). They were all unexpectedly downregulated (Figure 3B). Previous studies have shown that mycoparasitic species of Trichoderma activated chitinase when interacting with A. bisporus (Guthrie and Castle, 2006; Ihrmark et al., 2010), in contrast to the findings of this study. However, the chitinases of T. gamsii were all downregulated when interacting with Fusarium graminearum (Zapparata et al., 2021), which was consistent with the data of this study. This suggests that P. penicillatus could change the expression of chitinases based on demands of specific situations when infecting other Ascomycetes fungi. In this study, chitin synthase 1 was upregulated in P. penicillatus. One possible explanation for this upregulation is that these chitinases were involved in the cell wall remodeling during P. penicillatus growth. Their downregulation could result in strengthened cell walls during interaction. Another possible explanation is that when in contact with M. sextelata, P. penicillatus suppresses its secretion of fungal chitinases to avoid recognition and the induction of defense reactions in M. sextelata. Several proteases, including subtilase family protease, metalloprotease, and serine proteinase, were activated during the infection process of P. penicillatus (Figure 3C). These genes were reported to be involved in the regulation of cell wall degradation in plants and microorganisms (Feng et al., 2014; Schaller et al., 2018). In particular, the subtilase family proteases are good candidates for this function since they were reported to be induced during mycoparasitism before and during contact with the host in different Trichoderma species (Suárez et al., 2007; Seidl et al., 2009). This indicated that these CWDEs play an important role in the parasitism of P. penicillatus on morels.

M. sextelata activated a series of mechanisms to respond once it sensed the invasion of P. penicillatus. A chitin recognition protein and an allergen Asp f 15 precursor in M. sextelata were upregulated during both infection stages (Figure 7; Supplementary Table S8), which could activate the downstream immune response (Sanchez-Vallet et al., 2015). M. sextelata upregulated a caffeine-induced death protein 2-domain-containing protein and a putative apoptosis-inducing protein, as well as downregulating a cyclin (Figure 7; Supplementary Table S8); these genes are widely reported to promote immunity by initiating programmed cell death (Saiki et al., 2011; Qi and Zhang, 2019). We hypothesize that M. sextelata activates an apoptotic response to escape the proliferation of the pathogen when P. penicillatus invades.

A change in the reduction-oxidation status is one of the earliest responses detected when cells are attacked (Frederickson Matika and Loake, 2014). The reduction-oxidation process plays a key role in both the host and pathogen during the interaction of P. penicillatus and M. sextelata, while the regulatory mechanisms differ. The cytochrome P450 family genes in M. sextelata and P. penicillatus showed an opposite trend of expression. Six of the seven cytochrome P450 in P. penicillatus were downregulated, while all five in M. sextelata were upregulated (Figures 3D, 7). A previous study reported that ent-kaurene-derived diterpenoids act as a virulence factor interacting with the bacterium Xanthomonas oryzae pv. oryzicola and rice (Lu et al., 2015) and inhibited the growth of Staphylococcus aureus and Bacillus subtilis (Fatope et al., 2010). Two ent-kaurene oxidase-like proteins that are cytochrome P450s were downregulated in P. penicillatus, which could lead to increased synthesis of diterpenoid cytotoxins in P. penicillatus (Wang et al., 2012). Once the diterpenoid cytotoxins were secreted into M. sextelata cells, the pathogenicity of P. penicillatus to M. sextelata might be enhanced. Interestingly, one isotrichodermin C-15 hydroxylase gene was downregulated in P. penicillatus, while another was upregulated in M. sextelata. Previous study reported that blocking an isotrichodermin C-15 hydroxylase in Fusarium sporotrichioides resulted in the accumulation of trichothecenes, a virulence factor of Fusarium. spp. (McCormick and Hohn, 1997; Ward et al., 2002). This study also observed increased expression of CYPs superfamily genes in M. sextelata after infection with P. penicillatus, including benzoate 4-monooxygenase CYP-like protein and NADPH-P450 reductase. Benzoate 4-monooxygenase of ascomycete Cochliobolus lunatus was reported to be involved in detoxification of benzoic acid (BA), a key intermediate in metabolism of aromatic compounds in fungi, as well as the detoxification of phenolic compounds (Podobnik et al., 2008; Berne et al., 2012). NADPH-P450 reductase was reported to plays a central role in chemical detoxification and insecticide resistance in insects and fungi (Lian et al., 2011; Xu et al., 2021). This information suggests that these CYPs in M. sextelata might participated in the detoxification of toxic metabolites produced by P. penicillatus. We also identified two ABCB1 proteins that were upregulated in M. sextelata following infection with P. penicillatus (Figure 7). We hypothesize that M. sextelata could transport harmful substances secreted by P. penicillatus out of its cells by activating the expression of ABCB1. This has been confirmed by previous studies that ABCB1, a member of the ABC transporter family, plays an important physiological role in protecting the tissues from xenobiotics and endogenous metabolites (Roy et al., 2021).

In fungi, tyrosinases are generally associated with the formation and stability of spores, defense and virulence, and melanin production (Halaouli et al., 2006). Once the cells have been damaged, melanin produced by the reaction of tyrosinase with its substrate forms a melanin barrier to protect against the invasion of pathogenic bacteria or their toxins (Janusz et al., 2020). In this study, tyrosinase metabolism was the most enriched KEGG pathway in the infected M. sextelata (Figure 6). Five tyrosinase genes in M. sextelata were upregulated after infection by P. penicillatus (Figure 7). This indicated their importance in M. sextelata against infection with P. penicillatus.

Fungal laccase, a multicopper oxidase, is usually induced by a variety of phenolic compounds (Piscitelli et al., 2011) and was reported to have a wide range of functions, including defense against stressful conditions (Lakshmanan and Sadasivan, 2016; Chakraborty et al., 2020). One 1,4-benzoquinone reductase was upregulated in P. penicillatus (Figure 3B; Supplementary Table S4) and could be involved in the downstream synthesis of phenolic compounds. Interestingly, laccase-2 was the most upregulated gene in infected M. sextelata (Figure 7; Supplementary Table S6). A previous study found that the activity of laccase-2 enhanced the resistance of A. bisporus to the T. aggressivum toxin (Sjaarda et al., 2015), which is 3,4-dihydro-8-hydroxy-3-methyl isocoumarin that contains a phenolic hydroxyl and lactone ring (Krupke et al., 2003). This suggests that P. penicillatus could synthesize phenolic toxins and secrete them into M. sextelata, and M. sextelata could detoxify these phenolic toxins by activating laccase expression. However, further experiments are needed to verify this hypothesis.

During P. penicillatus infection of M. sextelata, cell wall-degrading enzymes, such as glucanases, mannanases, and proteases were probably secreted by P. penicillatus to degrade the cell wall of M. sextelata. M. sextelata triggered programmed cell death to prevent the excessive proliferation of P. penicillatus and synthesize cytochrome P450 and laccase to detoxify the P. penicillatus toxins. The melanin barrier formed by tyrosinase was also a possible immune pathway in M. sextelata. Although these possible regulatory pathways proposed in this study still need further verification, they provided a theoretical basis for researchers to breed WMD-resistant varieties and develop prevention and control methods for WMD.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

YY: conceptualization, funding acquisition, validation, investigation, data curation, visualization, writing – original draft preparation, and writing – review and editing. HT: funding acquisition, validation, and investigation. TL: investigation and data curation. LL: resources and investigation. JT: resources. WP: funding acquisition and supervision. All authors contributed to manuscript revision, read, and approved the submitted version.

This research was funded by National Science Foundation of China (NSFC31901119), Science and Technology Project of Sichuan Province (2021YFYZ0026), SAAS-International Cooperation Project (2021ZSSFGH04), Edible Fungus Innovation Team of Sichuan Province (SCCXTD-2021-7), and Special Fund for Talent Introduction and Training of SAAS (510000-01-114852).

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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.