Glycerol-3-Phosphate Shuttle Is Involved in Development and Virulence in the Rice Blast Fungus Pyricularia oryzae

Abstract

The glycerol-3-phosphate (G-3-P) shuttle is an important pathway for delivery of cytosolic reducing equivalents into mitochondrial oxidative phosphorylation, and plays essential physiological roles in yeast, plants, and animals. However, its role has been unclear in filamentous and pathogenic fungi. Here, we characterize the function of the G-3-P shuttle in Pyricularia oryzae by genetic and molecular analyses. In P. oryzae, a glycerol-3-phosphate dehydrogenase 1 (PoGpd1) is involved in NO production, conidiation, and utilization of several carbon sources (pyruvate, sodium acetate, glutamate, and glutamine). A glycerol-3-phosphate dehydrogenase 2 (PoGpd2) is essential for glycerol utilization and fungal development. Deletion of PoGPD2 led to delayed aerial hyphal formation, accelerated aerial hyphal collapse, and reduced conidiation on complete medium (CM) under a light–dark cycle. Aerial mycelial surface hydrophobicity to water and Tween 20 was decreased in ΔPogpd2. Melanin synthesis genes required for cell wall construction and two transcription factor genes (COS1 and CONx2) required for conidiation and/or aerial hyphal differentiation were down-regulated in the aerial mycelia of ΔPogpd2 and ΔPogpd1. Culturing under continuous dark could complement the defects of aerial hyphal differentiation of ΔPogpd2 observed in a light–dark cycle. Two light-sensitive protein genes (PoSIR2 encoding an NAD+-dependent deacetylase and TRX2 encoding a thioredoxin 2) were up-regulated in ΔPogpd2 cultured on CM medium in a light–dark cycle. ΔPogpd2 showed an increased intracellular NAD+/NADH ratio and total NAD content, and alteration of intracellular ATP production. Culturing on minimal medium also could restore aerial hyphal differentiation of ΔPogpd2, which is deficient on CM medium in a light–dark cycle. Two glutamate synthesis genes, GDH1 and PoGLT1, which synthesize glutamate coupled with oxidation of NADH to NAD+, were significantly up-regulated in ΔPogpd2 in a light–dark cycle. Moreover, deletion of PoGpd1 or PoGpd2 led to reduced virulence of conidia or hyphae on rice. The glycerol-3-phosphate shuttle is involved in cellular redox, fungal development, and virulence in P. oryzae.

Chemical ecology of an apex predator life cycle

“…This is shown through significantly decreased abundances of citrate in the Hour 12 and Hour 24 living insects compared to the uninfected insect and the Hour 24 dead insects. Although not significant, a similar trend is observed for two other TCA-related metabolites, malate and sn -glycerol-3-phosphate, which aids in NAD + regeneration through the glycerol phosphate shuttle, as well as NAD + , NADH, fumarate {Shi, 2018}. This could mean these metabolites were diverted for the immune response, given the differences between the living and dead insects.…”

Proteomic and Phenotypic Analyses of a Putative Glycerol-3-Phosphate Dehydrogenase Required for Virulence in Acidovorax citrulli

“…In P. aeruginosa, the analogous G3P dehydrogenase plays a role in producing virulence factors (Daniels et al, 2014). In addition, in the rice blast fungus, M. oryzae, conidia formation and disease development were affected by G3P dehydrogenase, and the G3P dehydrogenase knockout mutant was less virulent than the wild-type strain (Shi et al, 2018). Thus, the G3P dehydrogenase that is indispensable for glycerol production from glucose is an important component of virulence.…”

“…Further, G3P dehydrogenase plays an important role in the virulence of Pseudomonas aeruginosa (Daniels et al, 2014). In Magnaporthe oryzae, the deletion of G3P dehydrogenase had negative effects on fungal development and virulence (Shi et al, 2018). The function of G3P dehydrogenase in Colletotrichum gloeosporioides is also related to conidiation (Wei et al, 2004).…”

PoRal2 Is Involved in Appressorium Formation and Virulence via Pmk1 MAPK Pathways in the Rice Blast Fungus Pyricularia oryzae

“…For stress tests, each of the strains was cultured on minimal medium [MM, 10g/L D-Glucose, 6g/L NaNO 3 , 1.52g/L KH 2 PO 4 , 0.52g/L KCl, 0.52g/L MgSO 4 ∙7H 2 O, 0.1% (v/v) trace elements, 0.1% (v/v) vitamin solution] with 0.8M sucrose, 0.8M sorbitol, 0.5M NaCl, 20μg/ml Congo red, or 50μg/ml Calcofluor white (CFW) at 25°C ( Qu et al, 2020 ). To assay conidial germination and appressorium formation, 20μl spore suspensions (5×10 4 conidia/ml) were dropped onto hydrophobic coverslips and incubated at 22°C for 4h post-inoculation (hpi; conidial germination) and for 8 and 24hpi (appressorium formation; Shi et al, 2018 ). To observe cAMP’s role on appressorium formation, 10mM 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (8-Br-cAMP; Sigma, Japan) was added to spore suspensions which were dropped on hydrophobic or hydrophilic surfaces (GelBond) for 8 and 24hpi.…”