Potato is one of the most consumed crops in the world. However, its world yield is
severely affected by the Colorado potato beetle that is becoming resistant to existing
pesticides, as well as by the infections caused by the quickly mutating PVY virus. The
goal of our experiments was to test more ecological pesticidal alternatives against the
colorado potato beetle, as well as to analyze plant defense response against PVY.
Double stranded RNA has a great pesticidal potential because of its selectivity and its
natural ability to degrade quickly into harmless products. The pesticidal activity of
dsRNA targeting Mesh gene has been tested. Mesh gene has a role in formation of septate
junctions in the midgut epithelium of Colorado potato beetle and has been proven in the
past to be an efective pesticide against Western corn rootworm. Double stranded Mesh
RNA (dsMesh) was produced by inserting the Mesh gene into L4440 plasmid enabling
the production of dsRNA. The construct was used to transform E. coli with a knockout in
the dsRNA degrading enzyme. We prepared an extract of the transformed bacteria which
was then used in the feeding trials. Our research shows an effective pesticidal acitivity of
in vivo dsMesh against colorado potato beetle larvae, comparable to in vitro produced
dsMesh and the most commonly used pesticide against the Colorado potato beetle,
spinosad.
In the research of plant defense mechanisms against PVY, the influence of PVY´s known
silencing supressor HcPro on the plant transcription factor PTI5, that activates the
transcription of defense genes, was investigated. By PCR the HcPro-TFP phusion was
produced and afterwards inserted into a binary plant expression vector via Gateway
cloning approach. This contruct was used to transiently cotransform Nicotiana
benthamiana plants in combination with a plant vector containing PTI5 gene fused with
the yellow fluorescent protein. From our observation of the transformed leaves using
confocal microscopy, we came to the conclusion that HcPro does not trigger plants´
defense response via PTI5, for it does not cause its expression, stabilization or change of
localization. Furthermore, from the phenotypization of transiently transformed plants we
determined that HcPro does not trigger the formation of necrotic lesions, a common
consequence of hypersensitive response, via Nytbr defense gene in potato plants cv.
Rywal.
|
