Gene expression of ABC-transporters in the fungal biocontrol agent Clonostachys rosea in response to anti-fungal metabolites from Pseudomonas chlororaphis

Clonostachys rosea strain IK726 and Pseudomonas chlororaphis strain MA342 are two active biological control microbes against many seed and soil-borne plant diseases. A combination mixture between C. rosea IK726 and P. chlororaphis MA342 may provide additive or synergistic effects on the biocontrol ability of these microbes, due to complementing modes of action. However, it is not known if C. rosea IK726 can tolerate the antifungal metabolites produced by P. chlororaphis MA342. In the current study, a dual culture assay and mycelial dry weight measurements were used investigate the effect of antifungal metabolites from P. chlororaphis MA342 on the growth rate and biomass production of C. rosea IK726. Our result showed that C. rosea IK726 displayed a high level of resistance to the antifungal metabolites from P. chlororaphis MA342. ABC transporters are membrane-spanning proteins that are known as drug efflux pumps in many microbes. Due to the high number of ABC transporter genes in the C. rosea IK726 genome, it is suggested that these proteins may be involved in the antibiotic tolerance of C. rosea. Therefore, 13 C. rosea candidate ABC transporter genes were selected, and the gene expression patterns were monitored using quantitative reverse transcription PCR during the treatment of C. rosea IK726 with a culture filtrate from P. chlororaphis MA342. The transcript levels of putative multidrug resistance (MDR) gene ABC4267 and mitochondrial peptide transporter gene ABC4987 were induced during 2-6 hours after treatment with MA342 culture filtrate, compared to the non-treated control, but after 24 hours the expression decreased to normal levels. The putative siderophore transporter gene ABC3069 was induced after 2 and 24 hours of culture filtrate treatment. Expression levels of the putative pheromone transporter gene ABC2419 and the pleiotropic drug resistance (PDR) gene ABC3525 were induced after 2 hours. Expression of putative secondary metabolite transporter genes ABC592 and ABC3918, MDR gene ABC3260 and PDR gene ABC4345 were induced after 6 hours and then decreased to normal levels. Treatment with P. chlororaphis MA342 filtrate had no influence on the expression levels of the putative pheromone transporter gene ABC2210, the secondary metabolite transporter genes ABC3433 and ABC2700, or the PDR gene ABC3189. The induction of both PDR and MDR genes may be involved in an early stage of detoxification in C. rosea IK726. The up-regulation of the putative mitochondrial ABC4987 may be a response to an increased ATP consumption caused by the ATP-driven ABC transporters in C. rosea IK726. The induction of ABC3069 may be related to Fe limiting conditions in the MA342 culture filtrate. Induction of ABC592 and ABC3918 may imply that C. rosea secretes compounds that inhibit MA342 enzymes present in the culture filtrate. We conclude that C. rosea IK726 possess sufficient mechanisms to tolerate antifungal compounds from MA342. Certain ABC transporter genes related to different important cellular functions were significantly induced by the treatment with P. chlororaphis MA342 culture filtrate. This result indicates that ABC transporters may play a role in tolerance/detoxification mechanisms in C. rosea IK726.