In Planta Subcellular Localization of Alfalfa dwarf virus proteins

BEJERMAN NICOLAS; MANN, KRINPREET; DIETZGEN RALF

Workshop; 11th Australasian Plant Virology Workshop; 2014

Alfalfa dwarf virus (ADV), a new putativecytorhabdovirus, was detected in lucerne (alfalfa) crops distributed throughoutArgentina. Diseased plants hadshortened internodes, a bushy appearance, deformations, puckering, epinasty ofleaflet blades, vein enations, and varying sized papillae on the abaxialleaflet surfaces. The full-lengthnucleotide sequence of the genomic RNA of ADV was recently determined, whichrevealed that the antigenomic sequence of the negative-sense viral RNA has sevenopen reading frames (ORFs). The genome organization of ADV was similar to thatof Strawberry crinkle virus (SCV), amember of the genus Cytorhabdovirus:3?-N-P-P3-M-G-P6-L-5?, where N is the nucleocapsid protein gene, P theputative phosphoprotein gene, 3 and 6 are genes coding for proteins of unknownfunction, M the putative matrix protein gene, G the glycoprotein gene, and Lthe putative polymerase gene. Each ofthe ORFs found, except that encoding the polymerase gene, was amplified, clonedinto pDONR221 and sequenced. The pDONR clones were then recombined into the pSITE-2CAbinary vector for in planta subcellular localization. The complete ORFs wereexpressed as fusions to the C termini of the green fluorescent protein. pSITEderivatives were transformed into Agrobacteriumtumefaciens, which were infiltrated into leaves of Nicotiana Benthamiana.  Followinginfiltration, the leaves were examined using confocal laser scanning microscopybetween 40 and 50 h postinfiltration and subcellular localization of eachprotein was compared with that observed for Lettuce necrotic yellows virus(LNYV), which is also a member of the Cytorhabdovirusgenus. The N and G localized both in cell periphery and nuclear membrane, theP3, M and P6 localized to cell periphery. Despite a lack of predictable nuclearlocalization signals in P, this protein is targeted exclusively to nuclei. Thesubcellular localization of P, P3 and M were distinct from that of LNYV;whereas P6 is not present in LNYV. This study indicated that cytorhabdovirusesare diverse in where its proteins are localized.