Subtilisin-like Pr1 proteases marking the evolution of pathogenicity in a wide-spectrum insect-pathogenic fungus

Abstract

Subtilisin-like Pr1 proteases of insect-pathogenic fungi are a large family of extracellular cuticledegrading enzymes that presumably determine a capability of hyphal invasion into insect hemocoel through normal cuticle infection, but remain poorly understood although often considered as virulence factors for genetic improvement of fungal potential against pests. Here, we report that not all of 11 Pr1 family members necessarily function in Beauveria bassiana, an ancient widespectrum pathogen evolved insect pathogenicity~200 million years ago. These Pr1 proteases are phylogenetically similar to or distinct from 11 homologues (Pr1A-K) early named in Metarhizium anisopliae complex, a young entomopathogen lineage undergoing molecular evolution toward Pr1 diversification, and hence renamed Pr1A1/A2, Pr1B1-B3, Pr1 C, Pr1F1-F4,4 and Pr1 G, respectively. Multiple analyses of all single gene-deleted and rescued mutants led to the recognition of five conserved members (Pr1C, Pr1G, Pr1A2, Pr1B1, and Pr1B2) contributing significantly to the fungal pathogenicity to insect. The conserved Pr1 proteases were proven to function only in cuticle degradation, individually contribute 19-29% to virulence, but play no role in post-infection cellular events critical for fungal killing action. Six other Pr1 proteases were not functional at all in either cuticle degradation during host infection or virulence-related cellular events post-infection. Therefore, only the five conserved proteases are collectively required for, and hence mark evolution of, insect pathogenicity in B. bassiana. These findings provide the first referable base for insight into the evolution of Pr1 family members in different lineages of fungal insect pathogens.

Nuclear Ssr4 Is Required for the In Vitro and In Vivo Asexual Cycles and Global Gene Activity of Beauveria bassiana

“…Fungal lethal action against insects depends on not only an ability to infect the host by hyphal penetration through insect cuticle but also intrahemocoel transition of penetrating hyphae to unicellular blastospores for rapid proliferation by yeast-like budding until host death from mummification. In general, hyphal penetration through insect cuticle relies upon the cuticle-degrading activities of secreted enzymes, including Pr1 family proteases (22,51). In B. bassiana, pathogenicity abolished or greatly reduced via cuticle infection is often correlated with hindered secretion of such enzymes in the absence of one of some genes that function in different pathways, such as those encoding the Hsp40 family member Mas5 (23), the Na ϩ /H ϩ antiporter Nhx1 (24), the eisosome proteins Pil1A and Pil1B (25), the blue-light receptor VVD (27), and the histone acetyltransferase Gcn5 (26) or deacetylase Rpd3 (52).…”

“…First, conidial hydrophobicity as an important index of conidial adhesion to insect integument was assessed in a diphase (aqueous/organic) system as described previously (54,55). Second, total extracellular enzyme (ECE) and Pr1 activities essential for cuticle degradation were quantified from the supernatants (crude extracts) of 3-day-old submerged cultures in CDB (i.e., agar-free CDA) by reading optical densities at 442 nm (OD 442 ) and 410 nm (OD 410 ), respectively, as described elsewhere (23,26,51). The submerged cultures were initiated with 50-ml aliquots of a 10 6 conidia/ml suspension in CDB containing the sole nitrogen source of 0.3% BSA for enzyme induction.…”

P-type Na+/K+ ATPases essential and nonessential for cellular homeostasis and insect pathogenicity of Beauveria bassiana

“…Hyphal invasion into host insect requires secretion and action of extracellular (proteolytic, chitinolytic and lipolytic) enzymes including subtilisin-like Pr1 proteases to degrade and penetrate through the host cuticle [ 38 , 39 ]. After entry into the host hemocoel, penetrating hyphae turn into unicellular hyphal bodies (blastospores) for acceleration of fungal propagation by yeast-like budding until host mummification to death, at the time of which hyphal bodies turn back into the hyphae to penetrate again through the cuticle for outgrowth and conidiation on cadaver surfaces [ 40 , 41 ].…”

“…Exceptionally, deletion of ena1 in C. neoformans led to abolished pathogenicity to mice as well as increased cell sensitivity to alkaline pH [ 21 ]. In B. bassiana , essential roles of both ena1a and ena2b in normal host infection and insect pathogenicity are well indicated by an uncomputable LT 50 for their deletion mutants against the model insect via normal cuticle infection, a loss of their ability to grow out of cadaver surfaces by cuticular penetration, and great reductions in total ECE and Pr1 activities critial for cuticle degradation [ 38 , 39 ]. Since the deletion mutants were compromised slightly in conidial germination and not affected in hyphal growth on agar plates or in submerged cultures, we consider that the reduced ECE and Pr1 activities could be responsible for a loss of their pathogenicity and likely associated with blocked secretion of those enzymes due to the disturbance of their cellular homeostasis.…”

Three proline rotamases involved in calcium homeostasis play differential roles in stress tolerance, virulence and calcineurin regulation of Beauveria bassiana

“…Next, we quantified biomass level of each strain from the 3-day-old cultures of a 10 6 conidia/ml suspension in trehalose-peptone broth (TPB) mimicking insect hemolymph due to a speed of host death from mummification largely depending on proliferation of fungal cells in host hemocoel (Cai, Wang, Shao, Ying, & Feng, 2018;Gao, Mou, Tong, Ying, & Feng, 2020;Tong et al, 2018). As a result, biomass level decreased by 32% in Δfpr1, 38% in Δfpr2, and 85% in Δfpr3 compared to the WT strain (Figure 6c).…”