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To overcome plant resistance genes, viruses generally need to replicate under restrictive host conditions to incorporate adaptive genetic changes. The type, number, order, and speed of these viral changes impact plant resistance durability (3). Although most resistance (R) genes deployed against virus infections have lasted more than 25 years in the field (7), the effectiveness of Rz1, which confers partial resistance against Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania in sugar beet, has been compromised by the reemergence of the disease after 15 years of commercial field deployment of Rz1 in North America (18,19,27). The Benyvirus BNYVV is a multipartite, single-stranded, positive-sense RNA virus. RNA-1 and -2 encode the essential elements for replication, encapsidation, and cellular translocation, whereas RNA-3, -4, and isolate-specific, RNA-5, encode proteins involved in pathogenesis, vector transmission, and suppression of gene silencing (17,25,31). Despite its divided genome and the potential of mixed infections with different strains (13), high genetic stability seems to be the norm between spatiotemporally separated populations (11). In an otherwise genetically stable BNYVV genome, p25 (encoded by RNA-3) and p26 (RNA-5) genes are the most variable genomic regions, with strong positive selection acting on some of their codons (28). These genes operate synergistically to exacerbate symptoms in certain sugar beet cultivars (10,17), although p25 accounts for most rhizomania expression (32). Rhizomania is characterized by profuse lateral root proliferation, taproot constriction, root necrosis, and leaf chlorosis without foliar virus infection. The resistance mechanisms governed by Rz1 are phenotypically expressed by restricting virus accumulation in taproots and suppressing rhizomania development (32). At the biochemical level, resistance is associated with differential expression of genes involved in pathogenesis and hormonemediated plant development (4,15,29). Despite these plant defense responses, BNYVV still accumulates at low levels in asymptomatically infected roots of Rz1 plants. By reverse genetics, Koenig et al. (12) demonstrated, that for European A type isolates E12 and S8, valine at position 67 of the BNYVV p25 protein is required to overcome Rz1-mediated resistance and allow normal virus replication. This amino acid substitution was previously associated with breakdown of Rz1 in field-infected plants from the California Imperial Valley (CIV) (2). However, Liu and Lewellen (18) did not find a correlation between p25 sequences of numerous North American isolates and their titer in soil-inoculated Rz1 plants in greenhouse assays. This observation suggested that BNYVV might mutate in different ways to overcome Rz1 in North America. Therefore, the objective of this work was to explore the genetic diversity of the BNYVV p25 gene that might be associated with expression of rhizomania in Rz1 plants in the field.
MATERIALS AND METHODS
Field sampling. For initial investigation of BNYVV sequences, the virus was baited from field soil samples as described by Acosta-Leal and Rush (2). These infested soil samples, some of which had been collected as early as 1991, were from the rhizosphere of symptomatic and asymptomatic Rz1 plants, and symptomatic susceptible (rz1) plants from different sugar beet production regions around the United States. The name of these isolates provides information about their origin (i.e., field name, when it was known, state, and collection year). BNYVV rarely infects the aerial part of sugar beet plants but root-infected plants normally develop upright leaves with generalized chlorosis during maturation. This foliar symptom facilitates identification of plants with rhizomania, which frequently have a cluster distribution in the field (27). For virus quantification and genotyping using TaqManspecific probes, four to six Rz1 plants were collected from both inside and outside a yellow patch of plants with rhizomania and individually analyzed for virus infection. Each sample consisted of ≈۰٫۱ g of diseased hairy roots, or normal lateral roots from those plants without any root expression of the disease. Samples from Minnesota (MN) were from Crookston (four fields, 2005), Willmar (seven fields, 2005), and Roseland (one field, 2007), whereas samples from CIV were from four fields surveyed in 2006. Total RNA extractions. Root samples were collected in 2-ml microfuge tubes that contained a sterile 4-mm stainless steel grinding ball and then stored at –۸۰°C until processing. During RNA extraction, the plant tissue was first powdered by immersing the unopened 2-ml tubes containing the sample in liquid nitrogen and then immediately shaking them at 1,600 rpm for 2 min in a Talboys high-throughput homogenizer (Thorofare, NJ). Then, total RNA was extracted following the RNeasy Plant Mini Kit (Qiagen, Inc., Valencia, CA) protocol. All filtrations and filter drying were performed by centrifugation at 16,000 × g for 1 min at room temperature. This protocol usually yielded total RNA per sample at 200 to 500 ng µl–۱٫ Real-time reverse-transcription polymerase chain reaction viral RNA quantifications. The concentration of nucleic acids in total RNA-preparations was estimated by spectrophotometry and adjusted to 20 ng µl–۱ for viral RNA quantification. The amount of viral RNA encoding sequences recognized by specific TaqMan probes was estimated by relative quantification (RQ) real-time reverse-transcription polymerase chain reaction (RT-PCR) or directly by the cycle threshold (Ct) values generated without the incorporation of standards in the real-time RT-PCR reaction. RQ was calculated by the ΔΔCt method (20) using 18S ribosomal RNA as an endogenous reference (Applied Biosystems, Inc., Foster City, CA) and a plant RNA sample with the lowest detectable virus titer as the calibrator. This procedure determined the times a TaqMan-targeted RNA molecule was above the calibrator sample. To estimate the BNYVV RNA-2 titer, primers 50F (5′-CCGTTTTCCACAGACACTAACTATGTA-3′) and 51R (5′- TGCTAACCCTGAATCAGTTAAAGTACTT-3′) plus the TaqMan probe NYCP (6FAM-TGCACTTGTGTTATATGTTAATCTGTCTGACCCAG-TAMRA) were incorporated in one-step RTPCR to target the core of the coat protein (CP) gene (2). For detection and quantification of viral RNA-3 encoding specific sequences in the hypervariable coding region of p25, the allelic discrimination primers and 3′ minor groove binder TaqMan probes described by Acosta-Leal and Rush (2) were utilized. Real-time reactions were performed by an ABI Prism 7000 system (Applied Biosystems, Inc.) using the following sequential conditions: reverse transcription at 48°C for 30 min, reverse transcriptase inactivation at 95°C for 10 min, and amplification during 40 cycles of denaturing at 95°C for 15 s and annealing at 60°C for 1 min.
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