75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

301 Effects of cabbage leaf curl virus infection in Arabidopsis on small RNA pathways
Steven Bernacki, James Moyer, Niki Robertson
North Carolina State University
Cabbage leaf curl virus (CaLCuV) is a DNA geminivirus that causes severe symptoms in Arabidopsis. Removal
of the coat protein gene attenuates viral symptoms and allows the insertion of up to 800 nt of foreign DNA that can be
used to silence specific host genes by virus-induced gene silencing (VIGS). We previously demonstrated that RDR6 and
SGS3 are required for VIGS from CaLCuV (Muangsan et al., Plant J. 2004). Here, we analyze the role of Hen1 by using
CaLCuV to simultaneously silence Hen1 and ChlI, a visual marker for endogenous gene silencing. ChlI silencing was
reduced when Hen1 was simultaneously silenced, a result also found in rdr6 and sgs3 mutants. Unlike rdr6 and sgs3,
there was no increase in viral symptoms. The attenuation of ChlI silencing when ChlI and Hen1 were both inserted to the
CaLCuV vector could not be attributed to silencing two genes instead of one, because CaLCuV:GFP/ChlI-silenced plants
showed visible silencing similar to CaLCuV:ChlI-silenced controls. A relationship between viral symptoms and miRNA
pathways has been suggested because miRNAs are often upregulated in virus-infected plants. Similar to other RNA
viruses, CaLCuV-infected Arabidopsis plants show increased levels of Dcl1 and the miRNA that regulates it, miR162. This
increase was maintained even when CaLCuV was used as a silencing vector for ChlI. Surprisingly, when both Hen1 and
ChlI were silenced by CaLCuV, miR162 levels returned to normal. This could be due to the lack of stability of miR162
in the absence of methylation by Hen1. Because symptoms were not attenuated in CaLCuV:ChlI/Hen1-silenced plants
compared to CaLCuV:ChlI-silenced plants, our results raise the possibility that increased accumulation of at least some
miRNAs may not be directly related to viral symptom formation. This project was supported by Initiative for Future
Agriculture and Food Systems Grant no. 2001-52101-11507.
302 Characterization of Chromatin Surrounding Regions of Gene Expression within the
Arabidopsis Centromere
Andrew Cal, Song Luo, Daphne Preuss
University of Chicago
Despite a generally repressive heterochromatic environment, expressed genes have been localized to all 5 genetically
defined centromeres (Copenhaver et al 1999), and within the Cenp-A binding region of both rice centromere 8 (Nagaki et
al 2004) and a human neocentromere (Saffery et al 2003). We hope to gain understanding of how transcription proceeds
in the centromere by studying the chromatin environment surrounding centromeric regions of gene expression. DNA
methylation profiling by southern blot showed local hypomethylation within the regions. The expressed genes are also
associated with markers of open chromatin(acetylated histone H3, dimethylated histone H3 lysine 4) by ChIP assays,
and the heterochromatic mark dimethylH3 K9 is excluded from the hypomethylated regions, though it is sometimes
associated with flanking sequences. DNA methylation at the nucleotide level by bisulfite sequencing revealed distinct
methylation transitions 200-600 bp upstream from the transcriptional start site of all 5 genes examine. These sequences
are currently being tested for their ability to block heterochromatin spread.