Jayawardhane, K. | Search bioRxiv

bioRxiv2024

Functions of the AP2/ERF family transcription factor AIL7 in immunity against soilborne clubroot pathogen in Arabidopsis

Soilborne pathogens can be highly devastating, and clubroot, caused by Plasmodiophora brassicae, is particularly destructive to cruciferous plants. Although many AP2/ERF family transcription factors (TFs) have crucial physiological functions, very little is known regarding their functions in the context of soilborne diseases. Here we investigated the roles of AINTEGUMENTA-LIKE 7 (AIL7), an AIL sub-family TF in the AP2/ERF family, in plant immunity against clubroot. Unexpectedly, both AIL7 overexpression and mutant Arabidopsis lines exhibited increased tolerance to P. brassicae. Subsequent analysis revealed significant transcriptional alterations in genes linked to pathogen response, along with notable differences in genes associated with salicylic acid (SA) and jasmonic acid (JA) defense pathways, compared to wild-type plants. Interestingly, there was a tendency for up-regulation of SA- and JA-related genes in AIL7 overexpression and mutant lines in the absence, rather than presence, of P. brassicae. Subsequent phytohormone analyses confirmed these results. Taken together, AIL7 has an important role in maintaining constitutive systemic acquired resistance, involving phytohormone mediated defense, and this, rather than an accumulation of SA following P. brassicae challenge, primes the plants for improved clubroot resistance, which would shed light on exploring the functions of other AP2/ERF family TFs in plant immunity against soilborne pathogens.

pathology↗

bioRxiv2019

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl-CoA binding protein

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl-CoA-dependent biosynthesis of TAG and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over-expressed in some algal species, the detailed structure-function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure-function features of the hydrophilic N-terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N-terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N-terminal domains of animal and plant DGAT1s were previously found to bind acyl-CoAs, replacement of CzDGAT1 N-terminus by an acyl-CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N-terminus of the full-length CzDGAT1 could enhance the enzyme affinity for acyl-CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full-length CzDGAT1. Overall, our findings unravel the distinct features of the N-terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane-bound acyl-CoA-dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms. SIGNIFICANCE STATEMENTHere, we explored the N-terminus of a microalgal DGAT1, a membrane-bound enzyme determining oil biosynthesis, using in silico analysis, truncation mutagenesis, protein fusion and in vitro and in vivo characterization, and demonstrated its distinct structure-function features from the higher eukaryotic enzymes. We further engineered enhanced performance in DGAT1 via N-terminal fusion of ACBP, and obtained a kinetically improved enzyme with augmented protein production levels, which could boost oil accumulation in yeast and plant vegetative tissues.

plant biology↗