واکنش ژنوتیپ‌های سویای دارای آلل‌های ژن مقاومت (rsv1) به آلودگی با سه سویه از ویروس موزائیک سویا

ژن‌های مقاومت گیاه (r)، شناسایی هشدارهای (signals) غیر بیماری‌زایی (avirulent) مربوط به بیمارگرها را که منجر به فعال‌سازی پاسخ‌های دفاعی گیاه می‌شوند، برعهده دارند. از مدت‌ها پیش تصور بر این است که تحت‌فشار انتخاب، نرخ موتاسیون بالای ویروس‌های دارای ژنوم آران‌ای (rna)، به همراه اندازه بزرگ جمعیت و فاصله کوتاه تولید نسل، باعث ظهور جهش‌یافته‌هایی (mutants) از عوامل بیماری‌زا می‌شود که قادرند از شناسایی به‌وسیله ژن‌های r میزبان فرار کنند. در این مطالعه، پاتوسیستم soybean mosaic virus (smv) /rsv1 برای شناسایی این فرضیه بکار برده شد. ژنوتیپ‌های سویای دارای آلل‌های مقاومتrsv1 با عصاره دارای نتاج ویروسی همسانه عفونی سویه‌های n، g7 و g7d ویروس موزائیک سویا روی گیاهان سویای حساس، مایه‌زنی شدند. بعد از گذشت بیست و یک روز از مایه‌زنی، گیاهان فاقد علایم ویروسی توسط آزمون الیزای غیرمستقیم (indirect elisa) بررسی شدند. یکی از ده گیاه york و kwanggyo و دو گیاه از سی گیاه ogden مایه‌زنی شده با عصاره دارای نتاج ویروسی به‌دست‌آمده از تکثیر مولکول دی‌ان‌ای مکمل (cdna) سویه n ویروس موزائیک سویا روی گیاهان سویای حساس که در آزمایشگاه همسانه‌سازی شده بود (molecularly cloned smvn یا psmvn)، علایم ویروسی را نشان دادند و در آزمون الایزای غیر‌مستقیم، مثبت بودند. در مایه‌زنی مکانیکی انجام شده با استفاده از عصاره به‌دست‌آمده از این گیاهان، مجدداً ویروس به گیاهان york، kwanggyo و ogden منتقل شده و توانست با این گیاهان سازگار شود. بعد از انجام آزمون آرتی پی‌سی‌آر (rtpcr) و پی‌سی‌آر آشیانه‌ای (nested pcr) و تعیین ترادف طول کامل سیسترون‌های hcpro (helper componentprotease) و p3 ویروس، موقعیت جهش نقطه‌ای ایجادشده و اسیدآمینه رمز شده در اثر تغییر نوکلئوتید در نقطه جهش، با استفاده از نرم‌افزار mega7 مشخص شد. در گیاهان york و kwanggyo، جهش نقطه‌ای در سیسترون hcpro و به ترتیب در موقعیت‌های نوکلئوتیدی 1138t و 1210a و در گیاه ogden، در سیسترون p3 ویروس و در موقعیت نوکلئوتیدی 3079a مشاهده شد.

Reaction of Soybean Genotypes Containing Alleles of Rsv1-Resistance Gene Inoculated with Three Strain of Soybean Mosaic Virus

Introduction: Among legumes, soybean [Glycine max (L.) Merr.] is an important plant that grown throughout the world. There are many pathogens that reduce soybean yield. Plant viral diseases cause serious economic losses in many major crops by reducing yield and quality. Soybean mosaic virus (SMV) is amongst important pathogens that infect soybean. Plants use resistance genes against the invasive pathogens. The identification mechanism is based on geneforgene hypothesis. Plant resistance (R) genes direct recognition of pathogens harboring matching avirulent genes (signals) leading to activation of host defense responses. It has long been hypothesized that under selection pressure the infidelity of RNA virus replication together with large population size and short generation times results in emergence of variants (mutants) capable of evading Rmediated recognition. Four R gene including Rsv1, Rsv3, Rsv4 and newly Rsv5 have found in soybean operating against SMV.Materials and Methods: In this study, Rsv1/Soybean mosaic virus (SMV) pathosystem was used to investigate this hypothesis. Infectious cDNA clones of SMVN (pSMVN) and SMVG7 (pSMVG7) inoculated biolistically on Essex (rsv1) and SMVG7d (pSMVG7d) on Williams82 (rsv1) served as the sources of parental viruses. Sap containing viral progenies in 50 mM phosphate buffer, pH 7.0, derived from the infected tissues of biolistically inoculated Essex (rsv1) and Williams82 (rsv1) served as inoculum to mechanically inoculate carborundumdusted primary leaves of other soybean genotypes. Soybean genotypes containing alleles of Rsv1 were inoculated with SMV strains N, G7 and G7d. Soybean genotype Williams82 (rsv1), susceptible to all strains of SMV and Rsv1genotype soybean PI96983, Kwanggyo (Rsv1k), Marshal (Rsv1m), Ogden (Rsv1t), Raiden (Rsv1r), Suweon97 (Rsv1sk) and Touson50 (Rsv1n) were used in this study. All soybean seed were obtained from fieldgrown plants shown to be free of SMV by indexing. The inoculated plants were maintained in a growth chamber operating at 22°C with a photoperiod of 16h light and 8h dark. After twenty one days of postinoculation, plants were examined by indirect ELISA and RTPCR. Nested PCR was done and after Sanger sequencing of PCR products, results were analyzed by using Finch TV and MEGA7 software. Total RNA was isolated from top fullydeveloped systemically infected trifoliate leaves using an RNeasy Plant mini kit (Qiagen) as instructed by the manufacturer. RTPCR was done in the presence of Superscript reversetranscriptase III (Invitrogen) as instructed by the manufacturer. Nested PCR amplification of the entire HCpro and P3 cistrons was done using two pairs of primers, SMV239s / SMV3910a and SMV482s / SMV3840a, in the presence of EX Taq polymerase (Takara Bio). The resultant amplicons were purified with a QIAquickPCR Purification Kit (Qiagen) or MinElute® PCR Purification Kit (Qiagen) and sequenced using primers SMV1468s, SMV1614a, SMV2289s and SMV2916s. Sequencing was done at The University of Tennessee DNA Sequencing Facility.Results and Discussion: One out of ten York and Kwanggyo plants and two out of thirty Ogden plants inoculated with viral progeny derived from the reproduction of soybean mosaic virus strain N complementary DNA (cDNA) molecule on susceptible soybean plants, which was cloned in the laboratory (molecularly cloned SMVN or pSMVN) showed viral symptoms and were positive in indirect ELISA. In mechanical inoculation using the sap from these plants, the virus was again transferred to York, Kwanggyo and Ogden plants and subsequently was adapted to these plants. After performing RTPCR test and determining the fulllength sequence of the HCPro (Helper ComponentProtease) and P3 cistrons of the virus, the position of the point mutation and the amino acid encoded by the nucleotide exchange at the mutation point, using MEGA7 software, was identified. In York and Kwanggyo plants, a point mutation was observed in the HCPro whereas in the Ogden plant in the P3 cistron.Conclusion: The evolution of viruses is unavoidably linked to the evolution of their hosts. Due to the mutations in virus genome and the breakdown of resistance of various soybean genotypes, as well as the fact that the genome of the soybean mosaic virus is RNA type and the possibility of mutation is high, it can be a warning sign for plant breeders producing soybean plants with resistance genes to different strains of the virus. RNA viruses apply all known mechanisms of genetic variation to ensure their survival. Also, the intersection of resistant and susceptible genotypes and the study of the allele of resistance genes can be effective in producing resistant genotypes. The identities of different Rsv genes need to be revealed and the key components in SMV resistant signaling pathway need to be identified. So, a good breedingforresistance strategy would aim to develop cultivars with resistance against a wide range of strains of SMV. Transgenic soybean lines expressing part of the P3 and HCPro genes have been showed a stable and enhanced resistance to several strains of SMV and have the potential to significantly increase soybean yield.