Heterotrimeric G-Protein Signaling Is Required for Cellulose Degradation in Neurospora crassa

Abstract

The filamentous fungus Neurospora crassa decomposes lignocellulosic biomass to generate soluble sugars as carbon sources. In this study, we investigated a role for heterotrimeric G-protein signaling in cellulose degradation. Loss of the Gα subunit genes gna-1 and gna-3, the Gβ subunit genes gnb-1 and cpc-2, the Gγ gene gng-1, or the gene for downstream effector adenylyl cyclase (cr-1) resulted in loss of detectable cellulase activity. This defect was also observed in strains expressing a constitutively active version of gna-3 (gna-3Q208L). We found that GNA-1 levels are greatly reduced in Δgna-3, Δgnb-1, and Δgng-1 strains, likely contributing to cellulase defects in these genetic backgrounds. The observation that gna-3Q208L Δgnb-1 strains exhibit cellulase activity, despite greatly reduced levels of GNA-1 protein, is consistent with positive control of cellulase production by GNA-3 that is manifested in the absence of gnb-1. Expression patterns for five cellulase genes showed that Δgna-1, Δgnb-1, and Δgna-3 mutants produce less cellulase mRNA than the wild type, consistent with transcriptional regulation. Δcpc-2 mutants had wild-type levels of cellulase transcripts, suggesting posttranscriptional control. In contrast, results for Δcr-1 mutants support both transcriptional and posttranscriptional control of cellulase activity by cAMP signaling. Cellulase activity defects in Δgna-3 mutants were fully remediated by cAMP supplementation, consistent with GNA-3 operating upstream of cAMP signaling. In contrast, cAMP addition only partially corrected cellulase activity defects in Δgna-1 and Δgnb-1 mutants, suggesting participation of GNA-1 and GNB-1 in additional cAMP-independent pathways that control cellulase activity. IMPORTANCE Filamentous fungi are critical for the recycling of plant litter in the biosphere by degrading lignocellulosic biomass into simpler compounds for metabolism. Both saprophytic and pathogenic fungi utilize plant cell wall-degrading enzymes to liberate carbon for metabolism. Several studies have demonstrated a role for cellulase enzymes during infection of economically relevant crops by fungal pathogens. Especially in developing countries, severe plant disease means loss of entire crops, sometimes leading to starvation. In this study, we demonstrate that G-protein signaling is a key component of cellulase production. Therefore, understanding the role of G-protein signaling in the regulation of the unique metabolism of cellulose by these organisms can inform innovations in strain engineering of industrially relevant species for biofuel production and in combatting food shortages caused by plant pathogens.

Parallel proteomic and phosphoproteomic analyses reveal cellobiose‐dependent regulation of lignocellulase secretion in the filamentous fungus Neurospora crassa

“…Altogether, the results suggest that cellobiose regulates the early adaptation of N . crassa to cellulose at post‐transcriptional, translational, and post‐translational levels (Collier et al, 2020; Cziferszky et al, 2002; Li, Pang, et al, 2019; Lin et al, 2019; Liu et al, 2020). Regulatory roles of these newly identified phosphorylations in these processes need to be examined in further studies.…”

“…A recent study suggests that G‐protein signaling is required for cellulase production in N . crassa and positively regulates cellulase production via cAMP levels through adenylyl cyclase (CR1; Collier et al, 2020). We found that the effectors of the cAMP/cGMP signaling pathway, including CR1, PKAc1, and PRK2, were differentially phosphorylated in response to both cellobiose and Avicel (Table 1).…”

Regulator of G Protein Signaling Proteins Control Growth, Development and Cellulase Production in Neurospora crassa

“…Cultures were grown at 25 °C in constant light with shaking at 200 RPM for 3 days. Cultures were collected and centrifuged as described [ 21 ]. The pellets containing hyphal mats and residual Avicel were photographed.…”

“…Alterations in adenylyl cyclase activity and protein levels and the resulting effects on cAMP amount have been identified as downstream effects of G protein signaling in N. crassa [ 13 , 18 , 19 , 20 ]. Our group has recently demonstrated that five of the six predicted G protein subunit genes (including the Gα subunits gna-1 and gna-3 ) and adenylyl cyclase are required for production of detectable cellulase activity in N. crassa [ 21 ].…”

Metabolic engineering of Neurospora crassa for increasing carotenoids synthesis

“…It is non-toxic and is traditionally used for food production in south eastern Asia and is considered safe for other technical applications (Perkins and Davis, 2000) making it a suitable strain for synthetic biology. It also has an added advantage where it can grow on discarded lignocellular biomass for itself secreting cellulose and semi-cellulose degrading enzymes (Collier et al, 2020;Liu et al, 2020), and is more tolerant (Feldman et al, 2019;Tiwari et al, 2018) in active synthesized terpenoidal compounds such as steroidal or triterpenoid saponins which has a potential of reducing future production cost of active terpenoid compounds used in pharmaceutical or functional food industry. Neurosporaxanthin (NX) a carboxylic xanthophyll is the major carotenoid product of N. crassa which is usually accumulated with varying amounts of its neutral carotenoids (NC) (Hornero-Méndez et al, 2018).…”