SsCat2 encodes a catalase that is critical for the antioxidant response, QoI fungicide sensitivity, and pathogenicity of Sclerotinia sclerotiorum

Highlights

• SsCat2 contributes to the predominant catalase activity of S. sclerotiorum.

• SsCat2 is critical in dealing with the oxidative stress.

• SsCat2-deletion strains showed decreased sensitivity to QoI fungicides.

• Deletion of SsCat2 resulted in the high expression of alternative oxidase gene.

• SsCat2 is required for the virulence.

Abstract

Sclerotinia sclerotiorum is a destructive necrotrophic fungal pathogen with worldwide distribution. The metabolism of reactive oxygen species (ROS) is critical for the development and infection process of this economically important pathogen. Hydrogen peroxide (H₂O₂) is converted into water and dioxygen by catalases, which are major ROS scavengers in cells. Several genes have been predicted to encode the catalases of S. sclerotiorum, but the critical ones that function in the ROS stress response are still unknown. In this research, a catalase gene called SsCat2 was found to contribute to the predominant catalase activity at the stages of hyphae growth and sclerotial development. SsCat2 transcripts were induced under oxidative stress, and the target deletion of SsCat2 led to significant sensitivity to H₂O₂, suggesting that SsCat2 is critical in dealing with the oxidative stress. SsCat2-deletion strains were sensitive to hyperosmotic stresses and cell membrane-perturbing agents, suggesting impairment in cell integrity due to the inactivation of SsCat2. The expression of the alternative oxidase-encoding gene was upregulated in the SsCat2-deletion strains, which showed decreased sensitivity to QoI fungicides. SsCat2-deletion strains showed impaired virulence in different hosts, and more H₂O₂ accumulation was detected during the infect processes. In summary, these results indicate that SsCat2 encodes a catalase that is related to the oxidative stress response, QoI fungicide sensitivity, and pathogenicity of S. sclerotiorum.

Introduction

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide anions (O₂-), and hydroxyl radicals (OH) play important roles as secondary messengers in many intracellular signaling pathways (Ray et al., 2012). However, ROS can also oxidize any molecule in cells and lead to DNA damage, protein inactivation, lipid peroxidation, and eventually cell death (Heller and Tudzynski, 2011). To maintain low intracellular levels of ROS, cells usually use nonenzymatic and enzymatic defence systems to remove oxidants from solutions. The enzyme system mainly includes catalases, superoxide dismutases, peroxidases, glutathione peroxidases, peroxiredoxins, and thioredoxins, which constitute a complex antioxidant response (Aguirre et al., 2006, Segal and Wilson, 2018).

Hydrogen peroxise (H₂O₂) is converted into water (H₂O) and dioxygen (O₂) by catalases, which are major ROS scavengers in cells. The catalases are widely distributed in prokaryotic and eukaryotic organisms (Zamocky et al., 2008). In general, catalases can be divided into three types: I) catalase with large-size subunit (LSCs), II) catalase with small-size subunit (SSCs), and III) SSCs with an additional C-terminal domain (Hansberg et al., 2012). Thus far, several monofunctional catalases have been found in most fungi. Yeasts usually contain two SSCs, while filamentous ascomycetes have more catalases consisting of two LSCs and one to four SSCs, which accumulate and function differently in fungal growth and cell differentiation (Hansberg et al., 2012).

The oxidative burst is characterized by the transient production of ROS by plant cells and is an early event during plant-microbe interaction. High levels of ROS can be detected in plant tissues that are infected by biotrophic, hemibiotrophic, or necrotrophic fungi (Lyon et al., 2004). Handling external ROS efficiently is critical to the success of pathogen colonies. There is evidence that the catalase activity is associated with infection by fungal pathogens, but the different catalase genes may have disparate cellular functions (Keissar et al., 2002, Zhang et al., 2004). Some catalase genes have been reported to be involved in the response to oxidative stress from pathogenic fungi, including a monofuntional catalase-encoding gene CAT3 in Cochliobolus heterostrophus (Robbertse et al., 2003) and an extracellular catalase gene Bccat2 in Botrytis cinerea (Schouten et al., 2002). However, the disruption of the LSC catalase gene CATB in Magnaporthe grisea makes the strain more resistant to H₂O₂ (Skamnioti et al., 2007). Catalase genes also exhibit diverse roles in the virulence of fungi pathogens. Both the CAT3 knock-out mutant of C. heterostrophus and the Bccta2 knock-out mutant of B. cinerea show normal virulence toward their hosts, while an CATB mutant of M. grisea is much less pathogenic than the wild-type strain (Schouten et al., 2002, Robbertse et al., 2003, Skamnioti et al., 2007).

Sclerotinia sclerotiorum is an economically destructive fungal pathogen with worldwide distribution. Unlike many biotrophic fungal pathogens, external ROS might facilitate infection by necrotrophic pathogen (Govrin and Levine, 2000). Silencing of the membrane-localized NADPH oxidases of soybean resulted in reduced ROS levels and enhanced resistance to S. sclerotiorum (Ranjan et al., 2018). Effectively surviving stress caused by external ROS is important for infection by S. sclerotiorum, and several genes involved in the antioxidative response are critical for its pathogenicity (Xu and Chen, 2013, Yu et al., 2019, Zhang et al., 2019). Based on a close examination of the genome of S. sclerotiorum, seven genes were predicted to encode catalases, and insertional inactivation of Scat1 encoding LSC catalase resulted in an attenuation of pathogenicity but increased tolerance to H₂O₂ (Yarden et al., 2014). Thus, it is still unknown what catalase-encoding genes have critical functions in the ROS stress response of S. sclerotiorum.

A gene called SsCat2 (SS1G_00547) was predicted to encode an SSC catalase and was identified in S. sclerotiorum in this study. The main focus of the study is to investigate the biological role of SsCat2 via gene disruption. SsCat2-deletion strains showed abnormal sclerotial development, H₂O₂ modulation, and impaired virulence on different plants. Interestingly, the deletion strains showed decreased sensitivity to QoI fungicide through enhanced activation of the alternative oxidative pathway. Overall, this research suggests a critical role of SsCat2 in the ROS stress response, QoI fungicide sensitivity, and pathogenicity of S. sclerotiorum.