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|Title: ||Development of OMMV as a VIGS vector for plant protection|
|Authors: ||Varanda, Carla|
|Issue Date: ||2021|
|Publisher: ||Sociedade Portuguesa de Bioquímica|
|Citation: ||3) Varanda, C.M.R.; Félix, M.D.R.; Materatski, P.; Campos, M.D.; Patanita, M.; Marques, N.; Clara, M.I.; Nolasco, G. (2021). Development of OMMV as a VIGS vector for plant protection. XXI SPB – National Congress of Biochemistry. Évora, 14 a 16 de outubro|
|Abstract: ||Background Viruses are responsible for several important plant diseases, however,
they have also been used in biotechnology with different purposes. Virus induced gene
silencing (VIGS) allows specific silencing of foreign genes that can be inserted in a
viral vector and then inoculated in plants. When a sequence of a foreign gene is
introduced in a VIGS vector, the plant infected with this vector will be signalized to
target that foreign viral RNA and destroy it. In addition, plant defense mechanisms will
also target and destroy any homologous RNA, even if it is constitutively expressed by
the plant. The VIGS approach can also be used for plant protection purposes; for
example, if a fragment of the genome of a pathogenic virus is inserted in a VIGS vector,
the plant will destroy it and become protected against a possible further infection of that
virus. Several plant viruses have been used as VIGS vectors however, their large
genomes, their difficult manipulation and the reduced number of hosts they infect
restrain their use as vectors. The Alphanecrovirus Olive mild mosaic virus (OMMV) has
characteristics that place it as a very promising vector tool. Its small genome makes it
easy to manipulate, and it causes only mild systemic symptoms in a wide range of
crops, which will facilitate their manipulation into symptomless constructs and allow its
application to a high number of plants.
Methods An OMMV-based vector is being developed under the project
TOMVIRPROTECT. An infectious OMMV full length clone, available at our
laboratory (pUC_OMMVFL5), was manipulated to obtain a symptomless OMMV
strain. A single mutation at nt18 (C to A) of the OMMV p6, a silencing suppressor
protein, formed a STOP codon at this region, resulting in the gene knockout.
OMMVp6mutant was then manipulated to carry the GFP reporter gene in its 5’ and 3’
ends and in both sense and antisense directions to test silencing efficiency.
Results The new mutated OMMV genome (OMMVp6mutant) was inoculated onto
Nicotiana benthamiana indicator plants where it caused no visible symptoms but viral
accumulation levels similar to OMMV wild type, as well as a systemic presence.
Relative GFP mRNA accumulation level in 16 C plants (plants constitutively expressing
GFP) was the lowest when GFP was placed in the 3’end of OMMVp6mutant and in
antisense direction. A multiple cloning site was introduced in this region to facilitate
introduction of further fragments to be silenced.
Conclusions These transformations resulted in obtaining an efficient OMMV VIGS
vector, which is intended to become available for the control of many important viral
|Appears in Collections:||MED - Comunicações - Em Congressos Científicos Nacionais|
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