MaOpy2, a Transmembrane Protein, Is Involved in Stress Tolerances and Pathogenicity and Negatively Regulates Conidial Yield by Shifting the Conidiation Pattern in Metarhizium acridum.

Opy2 is an important membrane-anchored protein upstream of the HOG-MAPK signaling pathway and plays important roles in both the HOG-MAPK and Fus3/Kss1 MAPK. In this study, the roles of MaOpy2 in Metarhizium acridum were systematically elucidated. The results showed that the MaOpy2 disruption significantly reduced fungal tolerances to UV, heat shock and cell-wall-disrupting agents. Bioassays showed that the decreased fungal pathogenicity by topical inoculation mainly resulted from the impaired penetration ability. However, the growth ability of ∆MaOpy2 was enhanced in insect hemolymph. Importantly, MaOpy2 deletion could significantly increase the conidial yield of M. acridum by shifting the conidiation pattern from normal conidiation to microcycle conidiation on the 1/4SDAY medium. Sixty-two differentially expressed genes (DEGs) during the conidiation pattern shift, including 37 up-regulated genes and 25 down-regulated genes in ∆MaOpy2, were identified by RNA-seq. Further analysis revealed that some DEGs were related to conidiation and hyphal development. This study will provide not only the theoretical basis for elucidating the regulation mechanism for improving the conidial yield and quality in M. acridum but also theoretical guidance for the molecular improvement of entomopathogenic fungi.

MaSln1, a Conserved Histidine Protein Kinase, Contributes to Conidiation Pattern Shift Independent of the MAPK Pathway in Metarhizium acridum.

As a conserved sensor kinase in the HOG-MAPK pathway, Sln1 plays distinct functions in different fungi. In this study, the roles of MaSln1 in Metarhizium acridum were analyzed using gene knockout and rescue strategies. Deletion of MaSln1 did not affect conidial germination, conidial yield, or resistance to chemical agents. However, fungal tolerance to heat shock and UV-B were significantly reduced after deletion of MaSln1. Insect bioassays showed that fungal pathogenicity was significantly impaired when MaSln1 was deleted. Further studies showed that MaSln1 did not affect either germination or appressorium formation of M. acridum on locust wings, but it significantly increased appressorium turgor pressure. In addition, disruption of MaSln1 resulted in a conidiation pattern shift in M. acridum. Microscopic observation revealed, however, that some genes located in the MAPK signaling pathway, including MaSho1, MaHog1, MaMk1, and MaSlt2, were not involved in the conidiation pattern shift on SYA medium (microcycle medium). Meanwhile, of the 143 differently expressed genes (DEGs) identified by RNA-seq, no genes related to the MAPK pathway were found, suggesting that MaSln1 regulation of the conidiation pattern shift was probably independent of the conserved MAPK signaling pathway. It was found that 22 of the 98 known DEGs regulated by MaSln1 were involved in mycelial growth, cell division, and cytoskeleton formation, indicating that MaSln1 likely regulates the expression of genes related to cell division and morphogenesis, thus regulating the conidiation pattern shift in M. acridum. IMPORTANCE The productivity and quality of conidia are both crucial for mycopesticides. In this study, we systematically analyzed the roles of MaSln1 in fungal pathogens. Most importantly, our results revealed that deletion of MaSln1 resulted in a conidiation pattern shift in M. acridum. However, some other genes, located in the MAPK signaling pathway, were not involved in the conidiation pattern shift. RNA-seq revealed no genes related to the MAPK pathway, suggesting that the regulation of the conidiation pattern shift by MaSln1 was probably independent of the conserved MAPK signaling pathway. This study provided a new insight into the functions of Sln1 and laid a foundation for exploring the mechanisms of conidiation pattern shifts in M. acridum.

[Strain engineering and fermentation technology for production of long-chain dicarboxylic acid: a review].

Long-chain dicarboxylic acid (DCA), a building block for synthesizing a variety of high value-added chemicals, has been widely used in agriculture, chemical, and pharmaceutical industries. The global demand for DCA is increasing in recent years. Compared with chemical synthesis which requires harsh conditions and complicated processes, fermentative production of DCA has many unparalleled advantages, such as low cost and mild reaction conditions. In this review, we summarized the chemical and microbial synthesis methods for DCA and the commercialization status of the fermentation process. Moreover, the advances of using molecular and metabolic engineering to create high-yielding strains for efficient production of DCA were highlighted. Furthermore, the challenges remaining in the microbial fermentation process were also discussed. Finally, the perspectives for developing high titer DCA producing strains by synthetic biology were proposed.

O-mannosyltransferase MaPmt2 contributes to stress tolerance, cell wall integrity and virulence in Metarhizium acridum.

As a conserved post-translational modification, O-mannosyltransferase families play important roles in many cellular processes. Three subfamilies (MaPmt1, MaPmt2 and MaPmt4) are grouped in Metarhizium acridum according to sequence homology. The functions of MaPmt1 and MaPmt4 have been characterized in M. acridum previously. In this study, the functions of another member belonging to the Pmt2 subfamily, MaPmt2, were identified through RNAi strategy. The three MaPmt2 knockdown mutants showed dramatically decreased expression of MaPmt2. Phenotypic analyses showed that the mutants exhibited decreased tolerances to wet-heat, UV-B irradiation and cell wall perturbing chemicals. Further studies revealed that the mutants presented thinner cell walls observed by transmission electron microscope combined with changed cell wall components. Besides, knockdown of MaPmt2 decelerated conidial germination and decreased conidial yield. Compared with the wild-type strain, the MaPmt2 knockdown mutants caused impaired virulence only by topical inoculation. Results illustrated that the decreased virulence by inoculation could result from the delayed conidial germination on locust wings, reduced appressorium formation, as well as reduced turgor pressure in MaPmt2 knockdown mutants.

MaPmt1, a protein O-mannosyltransferase, contributes to virulence through governing the appressorium turgor pressure in Metarhizium acridum.

O-glycosylation is a very important post-translational modification of protein and involved in many cell processes in fungi. There exist three protein O-manosyltransferanse genes (MaPmt1, MaPmt2, MaPmt4) in Metarhizium acridum based on sequence homology. Here, MaPmt1, a gene for Pmt1 O-manosyltransferanse in M. acridum, was characterized and functionally analyzed through targeted gene disruption and complementation methods. Deletion of MaPmt1 had no effect on conidial germination, but slightly increased the conidial yield and significantly impaired fungal tolerances to UV-B radiation and wet-heat. Deletion of MaPmt1 made the fungus become more sensitive to cell wall disturbing agents and exhibit a thinner cell wall with changed components. Insect bioassays showed that disruption of MaPmt1 attenuated the fungal virulence significantly by topical inoculation but not by injection, indicating that MaPmt1 is required for penetration during the infection of M. acridum. Interestingly, deletion of MaPmt1 did not affect appressorium formation but significantly decreased appressorium turgor pressure. Moreover, the decreased virulence of MaPmt1 disruptant is mainly due to the reduced appressorium turgor pressure, which may be resulted from the declined glycerol concentration, combined with the weakened cell wall that could not hold the normal appressorium turgor pressure to penetrate the host cuticle.

The transmembrane protein MaSho1 negatively regulates conidial yield by shifting the conidiation pattern in Metarhizium acridum.

Sho1 is an important membrane sensor upstream of the HOG-MAPK signaling pathway, which plays critical roles in osmotic pressure response, growth, and virulence in fungi. Here, a Sho1 homolog (MaSho1), containing four transmembrane domains and one Src homology (SH3) domain, was characterized in Metarhizium acridum, a fungal pathogen of locusts. Targeted gene disruption of MaSho1 impaired cell wall integrity, virulence, and tolerances to UV-B and oxidative stresses, while none of them was affected when the SH3 domain was deleted. Intriguingly, disruption of MaSho1 significantly increased conidial yield, which was not affected in the SH3 domain mutant. Furthermore, it was found that deletion of MaSho1 led to microcycle conidiation of M. acridum on the normal conidiation medium. Deletion of MaSho1 significantly shortened the hyphal cells but had no effect on conidial germination. Digital gene expression profiling during conidiation indicated that differential expression of genes was associated with mycelial development, cell division, and differentiation between the wild type and the MaSho1 mutant. These data suggested that disruption of MaSho1 shifted the conidiation pattern by altering the transcription of genes to inhibit mycelial growth, thereby promoting the conidiation of M. acridum.