Functional Analysis of an Arabidopsis AAA+ ATPase in Syncytia

Transcriptome analysis of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots found that an AAA+ ATPase gene (At1g64110) was strongly expressed in syncytia. This gene is a member of a small gene family of three genes in Arabidopsis. Previous results have shown that this gene is important for syncytium development and abiotic stress responses. In this project we will further study the expression of this gene with antibodies. The subcellular location of the ATPases will be analysed using tags and antibodies. Since nothing is known today about the function of this ATPase, we will use T-DNA mutants and overexpression lines to address its possible function. AAA+ ATPases are widespread in plants and proteins with sequence similarity are also found in yeast. We will therefore use a complementation analysis of yeast mutants to find the function for this AAA+ ATPase. This should also shed light on the role of this protein in syncytia.

The Arabidopsis thaliana AAA+ATPase gene At1g64110, is a member of a small gene family which also includes At5g52882 and At4g28000. It was recently shown that At1g64110 is strongly expressed in syncytia induced by the cyst nematode Heterodera schachtii in A. thaliana roots. Artificial miRNA lines and T-DNA mutants were used to show that this ATPase is important for the development of syncytia. However, nothing is known yet about the function of these ATPases. A complementation analysis of Yeast mutants, defective in a range of AAA+ATPases, also did not give any hint about the possible function of the At1g64110 ATPase. In this study, constructs for recombinant expression of two short amino acid sequences derived from the At1g64110 AAA+ATPase were used to produce antibodies specific for this protein. The referring antigens where expressed in Escherichia coli, purified via HPLC and sent to an external service provider for the production of polyclonal antibodies in rabbits. Besides the specific antibodies also tag-specific antibodies against FLAG-tag and GFP where used to confirm the presence of the ATPase by Western blots. For this, constructs containing fusions of the ATPase with FLAG-tag and GFP were produced and transiently expressed in Nicotiana benthamiana for protein extraction and confocal laser scanning microscopy. These constructs and an overexpression construct without tag were also introduced into A. thaliana for the production of overexpression lines using the floral dip method. In addition, a triple knock-out mutant for all 3 ATPase genes and two promoter::GUS lines for At5g52882 and At4g28000 were produced. Nematode infection assays were carried out for quantitative analysis with knockout mutants and for GUS-staining of root tissue with the promoter::GUS lines. Proteins were extracted from different plant tissues for the immunoprecipitation of the At1g64110 ATPase followed by Western blot and mass spectrometry.