Related Experiment Videos
A gene for maackiain detoxification from a dispensable chromosome of Nectria haematococca
S F Covert1, J Enkerli, V P Miao
1Daniel B. Warnell School of Forest Resources, University of Georgia, Athens 30602-2152, USA.
Abstract:
In Nectria haematococca the MAK1 gene product converts a chick-pea (Cicer arietinum) phytoalexin, maackiain, into a less toxic compound. The presence of MAK1 in this fungal pathogen is also correlated with high virulence on chick-pea. Previous genetic analysis suggested that MAK1 is located on a meiotically unstable, dispensable chromosome. The unstable nature of this chromosome facilitated MAK1 cloning by allowing us to identify a subset of genomic cosmid clones likely to contain MAK1. Truncated forms of the chromosome, generated during meiosis, were isolated from strains either able (Mak+) or unable (Mak-) to metabolize maackiain and used to probe a chromosome-specific cosmid library. Only clones that hybridized exclusively to the chromosome from the Mak+ strain were then screened for their ability to transform a Mak- isolate to the Mak+ phenotype. A 2.7 kb HindIII-PstI fragment was subcloned from a cosmid conferring MAK1 activity, and its nucleotide sequence determined. Because MAK1 transcription is not induced strongly by maackiain, a reverse transcriptase-polymerase chain reaction was required to detect MAK1 transcription in a Mak+ strain, and to isolate MAK1 cDNA fragments. Comparison of the genomic and cDNA sequences of MAK1 revealed the presence of three introns and an open reading frame encoding a protein 460 amino acids in length. Two diagnostic domains in its deduced amino acid sequence suggest MAK1 encodes a flavin-containing mono-oxygenase. MAK1 is the first gene encoding maackiain detoxification to be cloned, and is the second functional gene cloned from this dispensable chromosome. Southern analysis of genomic DNA from ascospore isolates containing MAK2, MAK3, and MAK4 indicated that MAK1 is not homologous to other known maackianin-detoxifying genes.
Insights
The MAK1 gene in Nectria haematococca detoxifies a chickpea phytoalexin, maackiain, enhancing fungal virulence. Researchers cloned MAK1 from an unstable chromosome, revealing it encodes a flavin-containing monooxygenase.
Area of Science:
- Mycology
- Plant Pathology
- Molecular Biology
Background:
- Nectria haematococca utilizes the MAK1 gene to detoxify maackiain, a chickpea phytoalexin.
- MAK1 presence correlates with increased virulence in Nectria haematococca pathogens.
- Genetic analysis indicated MAK1 resides on a dispensable, meiotically unstable chromosome.
Purpose of the Study:
- To clone the MAK1 gene responsible for maackiain detoxification in Nectria haematococca.
- To characterize the MAK1 gene and its encoded protein.
- To understand the genetic basis of Nectria haematococca virulence on chickpea.
Main Methods:
- Utilized chromosome instability to identify MAK1-containing cosmid clones.
- Screened cosmid library using truncated chromosome fragments from maackiain-metabolizing (Mak+) and non-metabolizing (Mak-) strains.
- Employed reverse transcriptase-polymerase chain reaction (RT-PCR) to detect MAK1 transcription and isolate cDNA.
- Determined nucleotide sequence of the MAK1 gene and compared it to cDNA.
Main Results:
- Successfully cloned the MAK1 gene, conferring maackiain detoxification activity.
- Determined the nucleotide sequence of MAK1, revealing three introns and an open reading frame for a 460-amino acid protein.
- Deduced MAK1 encodes a flavin-containing monooxygenase based on conserved domains.
- MAK1 is the first gene for maackiain detoxification cloned and is not homologous to other known detoxification genes (MAK2, MAK3, MAK4).
Conclusions:
- The MAK1 gene is crucial for Nectria haematococca to detoxify maackiain, contributing to its virulence on chickpea.
- The cloning and characterization of MAK1 provide insights into fungal-plant interactions and detoxification mechanisms.
- MAK1 represents a novel functional gene from a dispensable fungal chromosome.