CRYOTHERAPY AS A METHOD FOR REDUCING THE VIRUS INFECTION OF APPLES (Malus sp.).

BACKGROUND: There is an urgent need in Kazakhstan for virus-free nursery stock to reinvigorate the industry and preserve historic cultivars. An in vitro collection of apples could be used for virus testing and elimination and to provide virus-free elite stock plants to nurseries. METHODS: Malus sieversii Ledeb. M. Roem. and Malus domestica Borkh. accessions were initiated in vitro for virus identification and elimination. Reverse transcription and multiplex PCR were used to test for five viruses. PVS2 vitrification was used as a tool for cryotherapy. RESULTS: Four viruses, Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) were detected in 17 accessions. Tomato ringspot virus (ToRSV) was not detected. ACLSV affected 53.8% of the accessions, ASPV 30.8%, ASGV 5.1%, and ApMV was found only in 'Aport Alexander'. Cryotherapy produced virus-free shoot tips for seven of nine cultivars tested. Six cultivars had 60-100% elimination of ACLSV. CONCLUSIONS: An in vitro collection of 59 accessions was established. Virus elimination using cryotherapy produced virus-free shoots for seven of nine cultivars and is a promising technique for developing a virus-free apple collection.

Phosphorylation of the alpha-subunit of plant eukaryotic initiation factor 2 prevents its association with polysomes but does not considerably suppress protein synthesis.

Phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and subsequent inhibition of protein synthesis is a major survival response to different stresses in animal and yeast cells. However, the role of this regulatory mechanism in plants is not unambiguously established to date. Here we describe a slight reduction of polysome abundance in Nicotiana benthamiana after the transient expression of a cDNA, AteIF2α(S56D), encoding a phosphomimetic form of Arabidopsis thaliana eIF2α. In contrast, the expression of a cDNA, AteIF2α(S56A), that encodes a non-phosphorylatable form of AteIF2α caused slightly elevated polysome formation compared to the control. Recombinant AteIF2α(S56A) was detected in association with 40S ribosomal subunit-containing complexes and also in the polysomal fraction, while recombinant AteIF2α(S56D) was detected mainly in complex with 40S subunits. Intentional phosphorylation of TaeIF2α induced by L-histidinol in a wheat germ (Triticum aestivum) cell-free extract did not reduce the abundance of polysomes. Interestingly, the phosphorylated TaeIF2(αP) was not detected in the polysomal fraction, similar to AteIF2α(S56D) in the in vivo experiment. Using mRNAs with a 'Strepto-tag' in the 3' untranslated region, the 48S pre-initiation complexes isolated from histidinol-treated wheat germ extracts were shown to contain phosphorylated TaeIF2(αP). Thus, the phosphorylation of plant eIF2 does not greatly affect its ability to participate in the initiation of mRNA translation, in contrast to animals and yeast, in which eIF2α phosphorylation results in profound suppression of protein synthesis.

Engineering the genome of Grapevine virus A into a vector for expression of proteins in herbaceous plants.

Grapevine virus A (GVA), a species of the genus Vitivirus, consists of a approximately 7.4 kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). In addition to grape varieties, GVA infects Nicotiana benthamiana plants and protoplasts. We engineered the genome of GVA as a vector that includes duplication of homologous sequences that contain the promoter of the movement protein (MP) sgRNA, supplemented by enzymatic restriction sites to be used as a convenient tool for transient expression of foreign genes from an individual sgRNA. The resulting vector was able to infect and to move in N. benthamiana plants in a manner similar to the wild-type GVA, but it was not stable and the inserted sequence was lost from the genome. Replacing the duplicated promoter with a GVA-MP promoter derived from a distantly related isolate of GVA improved the stability of the inserted sequence. The resulting vector was successfully used to express the reporter gene beta-glucuronidase (GUS) and the coat protein gene of Citrus tristeza virus in inoculated N. benthamiana plants. Development of a useful GVA vector is expected to find a use as a biotechnological tool for improvement of grapevines and it may enable vine breeders to bypass obstacles involved in genetic manipulation of perennial and fruiting plants.

ORF 5 of grapevine virus A encodes a nucleic acid-binding protein and affects pathogenesis.

A previous functional analysis of the genome of grapevine virus A (GVA) was not conclusive as to the role of open reading frame 5 (ORF 5). This ORF encodes a 10-kDa protein (p10) carrying two distinct domains: a basic, arginine-rich domain and a zinc-finger domain. P10 was cloned and expressed in Escherichia coli, and was shown by northwestern assays to interact with nucleic acids. In-frame deletion of the basic region abolished P10's nucleic acid-binding capability, whereas substitution of cysteine residues by serine in the zinc-finger domain did not affect binding. These mutations were inserted into the full-length infectious clone. It has been shown that ORF 5 mutations do not affect replication of GVA-RNA. However, plants inoculated with the aforementioned mutations did not develop symptoms, and Western blot analysis revealed markedly reduced expression of the movement protein (the product of ORF 3).

Two classes of subgenomic RNA of grapevine virus A produced by internal controller elements.

Grapevine virus A (GVA), a species of the recently established genus Vitivirus, consists of an approximately 7.3-kb single-stranded RNA genome of positive polarity, organized into five open reading frames (ORFs). The virus, which is closely associated with the grapevine rugose wood disease complex, has been poorly investigated genetically. We explored the production of viral RNAs in a GVA-infected Nicotiana benthamiana herbaceous host and characterized one nested set of three 5'-terminal sgRNAs of 5.1, 5.5, and 6.0 kb, and another, of three 3'-terminal sgRNAs of 2.2, 1.8, and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Neither 3'- nor 5'-terminal sgRNAs, which would correspond to ORF5, was detected, suggesting that expression of this ORF occurs via a bi- or polycistronic mRNA. The 5'-terminal sgRNAs were abundant in dsRNA-enriched extracts. Cloning and sequence analysis of the 3' end of 5.5-kb 5'-terminal sgRNA and the 5' end of the 1.8-kb 3'-terminal sgRNA suggested that a mechanism other than specific cleavage was involved in production of these sgRNAs. Apparently, the production of the 5'- and 3'-terminal sgRNAs was controlled by sequences upstream of the 5'-terminus of each of ORFs 2-4. Detection of both plus and minus strands of the 5'- and 3'-terminal sgRNAs, though in different levels of accumulation, suggested that each of these cis-acting elements is involved in production of four RNAs: a 3'-terminal plus-strand sgRNA which could act as an mRNA, the corresponding 3'-terminal minus-strand RNA, a 5'-terminal plus-strand sgRNA, and the corresponding 5'-terminal minus-strand RNA.