Roles of six Hsp70 genes in virulence, cell wall integrity, antioxidant activity and multiple stress tolerance of Beauveria bassiana

https://doi.org/10.1016/j.fgb.2020.103437Get rights and content

Highlights

  • Eight of 14 Hsp70 genes are essential for viability of Beauveria bassiana.

  • Six other genes (hsp70a-f) are dispensable for growth and conidiation.

  • Virulence is more compromised in the absence of hsp70f than of hsp70a-d.

  • The six genes are essential for cell wall integrity and antioxidant activity.

  • The six genes serve as differential sustainers of multiple stress tolerance.

Abstract

Functions of most Hsp70-coding genes remain unknown in filamentous fungi. Here, we show important roles of six Hsp70 homologs (Hsp70a-f) in Beauveria bassiana, a filamentous fungal insect pathogen that serves as a main source of wide-spectrum fungal insecticides. In phylogeny, Hsp70b-e, Hsp70g and Hsp70h are distinct from eight other partners that share the same clade with 14 Hsp70 proteins in model yeast. Among the 14 Hsp70-coding genes in B. bassiana, eight (hsp70g/hsp70h/ssa/ssb/ssc/kar/sse/ssz) were individually undeletable perhaps due to an essentiality of each for the fungal viability. Moderate defects in growth and conidiation occurred in the absence of hsp70e or hsp70f (lsh1). The disruption of hsp70f resulted in a reduction of 43% in conidial heat (45 °C) tolerance and of 97% in virulence, and the two reductions decreased to 19–27% and 18–45% in the disruption mutants of hsp70a to hsp70d respectively. Conidial UVB resistance decreased more in the absence of hsp70e (33%) than of hsp70f (25%), hsp70c (19%) or hsp70d (15%). All of these disruption mutants were differentially compromised in both cell wall integrity and antioxidant activity and also in cellular tolerance to Na+, Zn2+, Mn2+, Cu2+ and/or Fe2+. Delayed acidification of submerged cultures and reduced secretion of organic acids occurred only in the absence of hsp70b or hsp70f. Our findings indicate that hsp70a-f are important for both host infection and multiple stress tolerance of B. bassiana, and functionally similar to or beyond the lsh1, ssb and ssz homologs characterized previously in some plant-pathogenic fungi.

Introduction

Heat shock proteins (HSPs) constitute a large super family of evolutionarily conserved proteins classified to several families according to molecular sizes of less than 30 to ~100 kD and serve as molecular chaperones involved in an array of cellular processes and events (Li and Srivastava, 2004, Saibil, 2008). The families of Hsp60s (~60 kD), Hsp70s (~70 kD), Hsp90s (~90 kD) and Hsp100s (~100 kD) comprise most ATP-dependent HSPs and are critical for protein quality control under normal and stressful conditions (Verghese, Abrams, Wang, & Morano, 2012).

Members in the Hsp70 family are characterized by an HSP70 domain, a DnaK domain or both domains, and function as ubiquitous molecular chaperones in both prokaryotes and eukaryotes (Li & Srivastava, 2004). Multiple similar Hsp70 isoforms were speculated to be functionally similar in an organism but at least some of them have been shown to function in unique cellular events (Knighton et al., 2019, Lotz et al., 2019). In Saccharomyces cerevisiae, nine, three and two of 14 Hsp70s localize in cytoplasm, endoplasmic reticulum (ER) and mitochondria respectively (Boorstein, Ziegelhoffer, & Craig, 1994). The yeast Hsp70s feature an N-terminal ATPase domain, a central peptide-binding domain, and a variable C-terminal domain (Morano, Liu, & Thiele, 1998). Cytosolic Ssa and Ssb are important for posttranslational translocation (Craig, 2018, McClellan and Brodsky, 2000). The ER lumenal Kar2 is essential for cellular homeostasis and participates in the transport of nascent polypeptides into the ER lumen, polypeptide folding, and the recognition of misfolded proteins for degradation (Latterich and Schekman, 1994, Plemper et al., 1997). Lhs1, another ER lumenal HSP70 nonessential for yeast viability, is also involved in posttranslational translocation and repair of misfolded proteins in ER (Tyson & Stirling, 2000). Knockout mutation of Sse1 caused yeast growth defect (Mukai et al., 1993). More recent studies have revealed linkages of some yeast HSPs with mitogen-activated protein kinase (MAPK) pathways that regulate stress responses of different types. The revealed HSPs include an Hsp90 interacting with the MAPK Slt2 (Millson et al., 2005) or activating Slt2 via its induction by the HSP-specific transcription factor Hsf1 (Truman et al., 2007), Sse1 required for signaling through the cell-wall integrity pathway via partnership with Hsp90 and Slt2 (Shaner, Gibney, & Morano, 2008), and Fes1 acting as an Hsp70 nucleotide exchange factor essential for cell wall integrity (Kumar & Masison, 2019). In Candida albicans, Hsp90 was shown to coordinate a cross talk between the MAPK Hog1 and Slt2 (Mkc1) pathways (Leach et al., 2012). Among 13 Hsp70s (DK1-13) in Magnaporthe oryzae, MoSsb1 and MoSsz1 were shown to be crucial for growth, conidiation, pathogenicity and cell wall integrity (Yang et al., 2018), and MoLhs1 was evidently involved in the translocation of proteins across the ER membrane, the secretion of effector proteins and the activities of extracellular enzymes essential for host infection (Yi et al., 2009). In Fusarium graminearum, knockout mutants of FgSsb and FgSsz were compromised in mycelial growth and cell tolerances to carbendazim, heavy metal cations, high osmolarity and oxidation (Liu, Wang, Huang et al., 2017). Inactivation of FpLhs1 in Fusarium pseudograminearum also caused defects in hyphal growth, conidiation, conidial germination, and pathogenicity due to its role in protein secretion (Chen et al., 2019). An Hsp70 gene was preferentially expressed in Aspergillus nidulans under acidic conditions (Freitas et al., 2011) while inhibition of some Hsp70s by drugs in Aspergillus terreus increased antifungal amphotericin B resistance, which was not affected by overexpression of ssa and ssb (Blatzer et al., 2015). The previous studies demonstrate important roles of Hsp70s in yeast and filamentous fungi. In the large Hsp70 family, however, only a few members, such as Ssb, Ssz and Lhs1, have been shown to be crucial for filamentous fungal adaptation to host and environment, leaving it unknown whether and how many others function in filamentous fungi.

The roles for some Hsp70s in sustaining growth, conidiation, multiple stress tolerance and pathogenicity in plant-pathogenic fungi are of special merits for filamentous fungal insect pathogens, such as Beauveria bassiana and Metarhizium species serving as main sources of global mycoinsecticides and mycoacaricides (de Faria and Wraight, 2007, Wang and Wang, 2017). Fungal pesticides are composed of formulated cells as active ingredients, such as aerial conidia produced by solid fermentation or mycelia and blastospores produced in submerged cultures. Upon field application, so formulated fungal cells are inevitably exposed to outdoor stresses, such as persistently high temperatures close to or above their upper thermal limits, solar ultraviolet (UV) irradiation, and agrochemicals applied for controls of plant diseases and weeds. Such exposures may reduce the field stability and efficacy of fungal pesticides applied for arthropod pest control, making it necessary to understand molecular mechanisms involved in, and hence explore effective means to improving, multiple stress tolerance of formulated fungal cells (Tong and Feng, 2019, Tong and Feng, 2020, Ying and Feng, 2019, Zhang and Feng, 2018). Previously, two Hsp40 proteins, namely Mas5 and Mdj1, were shown to act as vital sustainers of conidiation capacity, insect pathogenicity and conidial heat tolerance as well as take part in cellular responses to UVB irradiation, high osmolarity, oxidation and/or cell wall perturbation and in transcription and translation of many phenotype-related genes in B. bassiana (Wang et al., 2016, Wang et al., 2017). These studies, though limited, demonstrate essential roles for HSPs in fungal adaptation to insect host and environment. Aside from Mas5 and Mdj1, however, many more HSPs have not been functionally characterized in fungal insect pathogens. This study sought to elucidate biological functions of six HSP70 proteins based on multiple phenotypic differences between single-gene disruption mutants and control (wild-type and complemented) strains.

Section snippets

Identification and bioinformatic analysis of Hsp70s in B. bassiana

The sequences of 14 Hsp70s in S. cerevisiae were used as queries to search through the B. bassiana genome under the NCBI accession NZ_ADAH00000000 (Xiao et al., 2012) via BLAST analysis (http://blast.ncbi.nlm.nih.gov/blast.cgi). Phylogenetic ties of all located homologs to the yeast queries and the counterparts in the NCBI protein databases of M. oryzae, F. graminearum, and F. pseudograminearum were analyzed with the neighbor-jointing method in MEGA7 software (http://www.megasoftware.net/).

Hsp70 family members essential and nonessential for viability of B. bassiana

Fourteen Hsp70 homologs were located in the B. bassiana genome with the yeast queries. Of those, seven share both HSP70 and DnaK domains and are highly conserved in S. cerevisiae and four examined filamentous fungi, including Ssa, Ssb, Kar, Ssc, Lhs, Sse and Ssz clustered to the same clade (Fig. 1A). This clade also contains the unique subclade Hsp70a found only in B. bassiana. Another distinct clade comprises six other homologs of B. bassiana, F. pseudograminearum and M. oryzae, and four of

Discussion

Among 14 Hsp70 homologs in B. bassiana, eight are classified to the same phylogenetic clade of all 14 S. cerevisiae Hsp70 proteins, including the unique Hsp70a that lacks homologs in other fungi examined, while the rest fall into a distinct clade comprising homologs of other filamentous fungi. This suggests greater difference in composition of the Hsp70 family between filamentous fungi and the model yeast than between filamentous fungal lineages. Our data provides the first insight into marked

CRediT authorship contribution statement

Jie Wang: Conceptualization, Methodology, Data curation, Writing - original draft, , Visualization, Investigation. Jianwen Chen: Visualization, Investigation. Yue Hu: Visualization, Investigation. Sheng-Hua Ying: Conceptualization, Methodology, Software, Validation. Ming-Guang Feng: Conceptualization, Methodology, Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Ying-Ying Huang (Core Facilities, School of Medicine, ZJU) and Jun-Ying Li (Analysis Center of Agrobiology and Environmental Sciences, ZJU) are acknowledged for technical assistance with flow cytometry and TEM analysis. This work was funded by the National Natural Science Foundation of China (31600060 and 31772218), the Ministry of Science and Technology of the People’s Republic of China (2017YFD0201202), Guangdong Province Science and Technology Innovation Strategy Special Fund (2018B020206001

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