The Maize An2 Gene is Induced by Fusarium Attack and Encodes an ent-Copalyl Diphosphate Synthase

Abstract

Using the technique of differential display, a maize transcript was identified whose silk tissue expression is induced in the presence of the ear rot pathogen Fusarium graminearum. The 3445 nt transcript includes a 727 nt 5' untranslated leader with the potential for extensive secondary structure and represents the maize gene An2. An2 encodes a copalyl diphosphate synthase (CPS)-like protein with 60% amino acid sequence identity with the maize An1 gene product involved in gibberellin (GA) biosynthesis. Recombinant expression and functional analysis demonstrated that both AN1 and AN2 are ent-copalyl diphosphate (ent-CPP) synthases (ent-CPS). Notably, the presence of an additional ent-CPS gene is consistent with previous reports that maize GA biosynthesis can proceed in the absence of An1. In addition, northern blot analysis showed that An2 transcript levels were strongly up-regulated by Fusarium attack, with an increase in silk, husk and ear tip tissues as early as 6 h after inoculation of silk channels with spore suspensions of various Fusarium sp. Gene expression of a third maize CPS-like gene, Cpsl1, is not affected by Fusarium infection. The Fusarium-inducible nature of An2 is also consistent with a previous report that cell-free extracts from maize seedlings produce ent-CPP derived diterpenes in response to Fusarium infection. However, it is not known whether An2 is involved in defense-related secondary metabolism in addition to GA synthesis.

Synergistic Substrate Inhibition of ent-Copalyl Diphosphate Synthase: A Potential Feed-Forward Inhibition Mechanism Limiting Gibberellin Metabolism

“…However, this does not necessarily lead to constructs suitable for overexpression. We have made equivalent truncations for other CPS enzymes, such as those we have previously identified from rice Xu et al, 2004) and maize (Harris et al, 2005) and, although we did see improvements in expression and/ or activity, the expression levels did not approach that observed with rAtCPS.…”

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

“…S7) and suggest a biosynthetic interconnectivity and potential biosynthetic competition between the kauralexin and dolabralexin pathways. ZmAN2 was first described in a differential display analysis of highly elicited transcripts following F. graminearum challenge of maize silks (Harris et al, 2005). Following the first description of ZmAN2, pathogen-elicited transcripts have been observed and reported in a large number of maize studies with diverse microbes Schmelz et al, 2011Schmelz et al, , 2014van der Linde et al, 2011;Vaughan et al, 2014;Christie et al, 2017).…”

“…S1), which differs from the clustering of genes of specialized diterpenoid pathways in rice . However, the maize ent-CPP synthases, ZmAN1, which functions in GA biosynthesis (Bensen et al, 1995), and ZmAN2, which has functions in specialized metabolism (Harris et al, 2005), colocalize with ZmKSL4 on chromosome 1, with distances of ;4 and ;159 Mb, respectively.…”

“…Circles indicate bootstrap support of greater than 80% (500 repetitions), and the functional association with the biosynthesis of ent-kaurene (yellow) or specialized diterpenoids (red) is highlighted. B, Total ion chromatograms (TIC) of reaction products resulting from E. coli coexpression assays of ZmKSL4 with ZmAN2 CPP synthase (Harris et al, 2005), rice CPS4 syn-CPP synthase , and the grand fir abietadiene synthase variant D621A producing (+)-CPP (Peters and Croteau, 2002) as compared with available authentic standards for product identification. Compound c represents a contamination product from the engineered E. coli system.…”

“…However, the frequently observed substrate promiscuity of characterized enzymes (Peters, 2010;Morrone et al, 2011;Zerbe and Bohlmann, 2015) also may enable roles in defense-related specialized metabolism. In the absence of mutant analyses, the maize kauralexin and zealexin metabolic pathways have remained largely unproven, with the exception of the ent-CPP synthase ZmAN2, which has been empirically demonstrated to function in kauralexin formation (Harris et al, 2005;Vaughan et al, 2015). The b-macrocarpene synthases ZmTPS6, ZmTPS11, and ZmCYP71Z18 have been assigned to the zealexin pathway based on predictions from in vivo biochemical assays (Köllner et al, 2008b;Mao et al, 2016).…”