<it>In planta </it>transient expression as a system for genetic and biochemical analyses of chlorophyll biosynthesis

• Main Authors: Sawers Ruairidh JH, Farmer Phyllis R, Moffett Peter, Brutnell Thomas P
• Format: Article
• Language: English
• Published: BMC 2006-09-01
• Series: Plant Methods
• Online Access: http://www.plantmethods.com/content/2/1/15

<p>Abstract</p> <p>Background</p> <p>Mg chelatase is a multi-subunit enzyme that catalyses the first committed step of chlorophyll biosynthesis. Studies in higher plants and algae indicate that the Mg chelatase reaction product, Mg-protoporphyrin IX plays an essential role in nuclear-plastid interactions. A number of Mg chelatase mutants have been isolated from higher plants, including semi-dominant alleles of <it>ChlI</it>, the gene encoding the I subunit of the enzyme. To investigate the function of higher plant CHLI, bacterial orthologues have been engineered to carry analogous amino acid substitutions to the higher plant mutations and the phenotypes examined through <it>in vitro </it>characterization of heterologously produced proteins. Here, we demonstrate the utility of a transient expression system in <it>Nicotiana benthamiana </it>for rapidly assaying mutant variants of the maize CHLI protein <it>in vivo</it>.</p> <p>Results</p> <p>Transient expression of mutant maize <it>ChlI </it>alleles in <it>N. benthamiana </it>resulted in the formation of chlorotic lesions within 4 d of inoculation. Immunoblot analyses confirmed the accumulation of maize CHLI protein suggesting that the chlorosis observed resulted from an interaction between maize CHLI and endogenous components of the <it>N. benthamiana </it>chlorophyll biosynthetic pathway. On the basis of this assay, PCR-based cloning techniques were used to rapidly recombine polymorphisms present in the alleles studied allowing confirmation of causative lesions. A PCR-based mutagenesis was conducted and clones assayed by transient expression. A number of novel allelic variants of maize Zm<it>ChlI </it>were generated and analyzed using this assay, demonstrating the utility of this technique for fine mapping.</p> <p>Conclusion</p> <p>Transient expression provides a convenient, high-throughput, qualitative assay for functional variation in the CHLI protein. Furthermore, we suggest that the approach used here would be applicable to the analysis of other plastid-localized proteins where gain-of-function mutations will result in readily observable mutant phenotypes.</p>