Robertson LAB
Research
Viral vectors carrying a host-derived gene fragment can induce silencing of the corresponding gene(s) in infected plants. We developed vectors from DNA viruses for the purpose of down-regulating genes involved in plant growth and development. Geminiviruses replicate in plant nuclei as double stranded DNA molecules and are therefore easy to work with. Geminiviruses had been developed for expression of genes in plants but have an upper size limit of about 800 bp, smaller than most plant coding sequences. Our work demonstrated that DNA viruses could be used to inhibit gene expression even though their genes are transcribed by RNA polymerase II and polyadenylated, as are nuclear genes. How geminiviruses themselves avoid host silencing machinery, when they replicate to thousands of copies in plant nuclei, remains an enigma.
We developed silencing vectors derived from two members of the Begomovirus genus of Geminiviridae: Tomato Golden Mosaic Virus (TGMV) and Cabbage Leaf Curl Virus (CaLCuV). Both viruses were engineered to facilitate insertion of plant gene sequences, 100 - 800 bp in size, without compromising viral replication or spread. Some geminiviruses require a coat protein for movement and can accept only about 100 bp of extra sequence. We showed that extensive silencing could result from such small sequences and that fragments from different genes cloned in tandem or in different genome components could produce silencing of multiple genes. These results suggest that geminiviruses, which infect many economically valuable plants, may be a useful tool for discovering or verifying gene function.
TGMV and CaLCV both have two components, designated A and B, which are similar in size. The common region (CR) is indicated by black boxes. The CR contains the viral origin of DNA replication and is conserved between the A and B components, but varies between members of the begomoviruses. The A component encodes genes needed for replication, gene expression, and encapsidation. AL1 is also known as AC1(required for replication), AL2 as AC2, and AL3 as AC3. The B component contains two proteins needed for viral movement, BR1 (BV1) and BL1 (BC1).
Last updated August, 2004
N. Muangsan (top) Charles Peele (bottom)
TGMV-mediated silencing in N. benthamiana
N. benthamiana 4-week old seedlings were co-bombarded with TGMV A and a TGMV B vector carrying a 154-bp fragment of SUcDNA. The tobacco SU gene encodes the I subunit of magnesium chelatase (ChlI ortholog), needed for chlorophyll formation. Yellow spots (red arrow) occur on bombarded leaves about 5 days after bombardment and then silencing spreads into new growth. Plant on left, 12 d.p.i., plant on right, 56 d.p.i. The plants do not lose SU silencing in new growth.
N. Munagsan
CaLCV-mediated
silencing in Arabidopsis
Arabidopsis Col0 were biolistically bombarded with a combination of
CaLCV A::ChlI (pMTCbLCVA.008) and pCPCbLCVB.002. Clorata42 (now known
as Chl1) encodes one of three subunits of magnesium chelatase,
an enzyme required for chlorophyll biosynthesis. The loss of endogenous
gene expression is seen as yellow-white regions of leaves produced after
inoculation. Photograph was taken at 20 days after bombardment. Uninfected
and inoculated leaves remain green.