Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi.

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.

Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen.

Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiae Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (∼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2 /M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen.

Insight into the transcriptional regulation of Msn2 required for conidiation, multi-stress responses and virulence of two entomopathogenic fungi.

Msn2/4 transcription factors in some fungi have null effects on virulence and cellular stress responses. Here we found that the transcriptional regulation of Msn2 orthologs is vital for the conidiation, virulence and multi-stress responses of Beauveria bassiana (Bb) and Metarhizium robertsii (Mr), which lack Msn4 orthologs. Compared to wild-type and complemented strains of each fungus with all similar phenotypes, ΔBbmsn2 and ΔMrmsn2 showed remarkable defects in conidial yield (∼40% decrease) and virulence (∼25% decrease). Both delta mutants lost 20-65% of their tolerances to hyperosmolarity, oxidation, carbendazim, cell wall perturbing and high temperature at 34 °C during colony growth. Their conidia were also significantly (18-41%) less tolerant to oxidation, hyperosmolarity, wet-heat stress at 45 °C and UV-B irradiation. Accompanied with the defective phenotypes, several conidiation- and virulence-associated genes were greatly repressed in ΔBbmsn2 and ΔMrmsn2. Moreover, differentially expressed genes in the transcriptomes of ΔBbmsn2 versus wild type were ∼3% more under oxidative stress, but ∼12% fewer under heat shock, than those in the ΔMrmsn2 counterparts. Many stress-responsive effector genes and cellular signaling factors were remarkably downregulated. Taken together, the two entomopathogens could have evolved somewhat distinct stress-responsive mechanisms finely tuned by Msn2, highlighting the biological significance of Msn2 orthologs for filamentous fungi.

Evidence for an ancient whole-genome duplication event in rice and other cereals.

Gene duplication has been proposed as an accelerator of evolution. Ancient genome duplication events have been identified in diverse organisms, such as yeast, vertebrates, and Arabidopsis. Here, we have identified a whole genome duplication event (WGD) in the rice genome, which took place prior to the divergence of grasses about 70 million years ago (mya). A total of 117 duplicated blocks were detected, which are distributed on all 12 chromosomes and cover about 60% of the rice genome. About 20% genes on these duplicated segments are retained as duplicate pairs. In contrast, 60% of the transcription factor genes are retained as duplicates. The identification of a WGD in the ancestral grass genome will impact the study of grass genome evolution, and suggest that polyploidization and subsequent gene losses and chromosomal rearrangements have played an important role in the diversification of grasses.