Identification of polyketide synthase genes required for aspinolide biosynthesis in Trichoderma arundinaceum

Abstract

The fungus Trichoderma arundinaceum exhibits biological control activity against crop diseases caused by other fungi. Two mechanisms that likely contribute to this activity are upregulation of plant defenses and production of two types of antifungal secondary metabolites: the sesquiterpenoid harzianum A (HA) and the polyketide-derived aspinolides. The goal of the current study was to identify aspinolide biosynthetic genes as part of an effort to understand how these metabolites contribute to the biological control activity of T. arundinaceum. Comparative genomics identified two polyketide synthase genes (asp1 and asp2) that occur in T. arundinaceum and Aspergillus ochraceus, which also produces aspinolides. Gene deletion and biochemical analyses in T. arundinaceum indicated that both genes are required for aspinolide production: asp2 for formation of a 10-member lactone ring and asp1 for formation of a butenoyl subsituent at position 8 of the lactone ring. Gene expression and comparative genomics analyses indicated that asp1 and asp2 are located within a gene cluster that occurs in both T. arundinaceum and A. ochraceus. A survey of genome sequences representing 35 phylogenetically diverse Trichoderma species revealed that intact homologs of the cluster occurred in only two other species, which also produced aspinolides. An asp2 mutant inhibited fungal growth more than the wild type, but an asp1 mutant did not, and the greater inhibition by the asp2 mutant coincided with increased HA production. These findings indicate that asp1 and asp2 are aspinolide biosynthetic genes and that loss of either aspinolide or HA production in T. arundinaceum can be accompanied by increased production of the other metabolite(s). Key points • Two polyketide synthase genes are required for aspinolide biosynthesis. • Blocking aspinolide production increases production of the terpenoid harzianum A. • Aspinolides and harzianum A act redundantly in antibiosis of T. arundinaceum.

Draft Genome Sequence of Aspergillus ochraceus Strain DY1, a Lignin-Degrading Fungus Isolated from Wood Rot

“…The draft genome sequence is 35,149,223 bp long, assembled into 211 contigs, with an N 50 value of 1,168,992 bp. The genome size is similar to the reported sizes of other Aspergillus ochraceus genome assemblies ( 5 , 6 ). Gene prediction and annotation were performed using the AUGUSTUS tool ( 7 ).…”

Effects of trichothecene production by Trichoderma arundinaceum isolates from bean-field soils on the defense response, growth and development of bean plants (Phaseolus vulgaris)

“…When grown at 20°C, TP6.6 produced a much lower level of trichodermol but higher levels of the polyketides aspinolides B and C ( Figure 3B ). In previous studies of T. arundinaceum , aspinolide production increased when HA production was blocked by inactivation of trichothecene biosynthetic genes or when conditions for HA production were not optimal ( Malmierca et al., 2015 ; Izquierdo-Bueno et al., 2018 ; Cardoza et al., 2019 ; 2022 ).…”

“…In addition, aspinolides B and C were found in T. arundinaceum TP6.6, but only when grown at 20°C. Production of aspinolides by T. arundinaceum was already reported ( Malmierca et al., 2015 ; Izquierdo-Bueno et al., 2018 ; Cardoza et al., 2022 ), and their production together with other additional aspinolides was observed mainly when production of HA was partial or totally blocked ( Malmierca et al., 2015 ; Izquierdo-Bueno et al., 2018 ). In addition, data observed in the present article also support the hypothesis that production of aspinolides is stimulated when T. aundinaceum was grown at suboptimal conditions, e.g., 20°C, as was previously described for IBT 40837 strain ( Cardoza et al., 2022 ).…”

“…Production of aspinolides by T. arundinaceum was already reported (Malmierca et al, 2015;Izquierdo-Bueno et al, 2018;Cardoza et al, 2022), and their production together with other additional aspinolides was observed mainly when production of HA was partial or totally blocked (Malmierca et al, 2015;Izquierdo-Bueno et al, 2018). In addition, data observed in the present article also support the hypothesis that production of aspinolides is stimulated when T. aundinaceum was grown at suboptimal conditions, e.g., 20°C, as was previously described for IBT 40837 strain (Cardoza et al, 2022). Furthermore, in correlation with their ability to produce HA, the three isolates also shown a significant antifungal activity against the bean pathogens R. solani and S. sclerotiorum, in comparison to the HA non-producer mutant Dtri5.3, which agrees with previous reports indicating the importance of HA production on the antifungal activity of the producer Trichoderma isolates (Malmierca et al, 2013).…”

Endophyte genomes support greater metabolic gene cluster diversity compared with non-endophytes inTrichoderma

“…BGC repertoire diversification in Trichoderma is in part shaped by horizontal gene transfer between Eurotiales, Hypocreales, and Xylariales. Given the precedent of horizontal transfer in Hypocreales and Brevicompactum clade [131–133], we were not entirely surprised to identify BGC acquisitions among Trichoderma species, including the ergot alkaloid BGC [134]. The species phylogeny places Clavicipitaceae and Trichoderma within Hypocreales.…”