| Barley yellow dwarf viruses(BYDVs) are different viruses in the family Luteoviridae, only transmitted by wheat aphids in a persistent, circulative, non-propagative manner. BYDV-GAV is a special BYDV only reported in China, and it has become the most common pathogen of wheat yellow dwarf disease, causing severe yield losses in wheat production. Breeding and planting of resistant varieties is the most effective and economical method to control wheat yellow dwarf disease. However, there are few BYDVs-resistant wheat varieties being used widely in practical application, with resistance genes from limited origins. Screening of resistant sources to control wheat yellow dwarf disease is a problem to be settled urgently. At the same time, relatively few studies focused on the population genetics, functions of genes or molecular pathogenic mechanism for BYDV-GAV. Therefore, wheat yellow dwarf disease caused by BYDV-GAV was researched from two aspects of host resistance and virus pathogenicity in this study. The main results were as follows:1. It was demonstrated that Psathyrostachys huashanica, a wheat-relative wild species native to China, could be infected by BYDV-GAV after inoculation by aphids in greenhouse. Virus particles could be transmitted from P. huashanica back to healthy wheat by virus-free aphids. RT-PCR and TAS-ELISA were used for BYDV-GAV detection from inoculated and non-inoculated leaves of tested plants at regular intervals after inoculation. The results demonstrated that BYDV-GAV propagates slowly in P. huashanica with a lower virus load and the systematic movement in P. huashanica is suppressed. In another word, P. huashanica is highly resistant to BYDV-GAV. The BYDV-GAV resistance for 31 wheat-P. huashanica hybrid lines were assessed in greenhouse and field in three consecutive years. A total of six lines(H9020, H9020-1-6-8-3, H9020-17-25-6-4, H9014-27-1-1-13-5-5, H9020-17-5, H9014-157-2-15-3) showed lower virus load compared with their female parent wheat 7182 after 21 days post inoculation in greenhouse. A total of four lines(H9020, H9020-1-6-8-3, H9020-17-25-6-4, H9015-17-4-4) showed milder symptoms and less loss of TKW(Thousand Kernel Weight) compared with their female parent wheat 7182. The results indicated that partial wheat-P. huashanica hybrid lines are resistant to BYDV-GAV, and the BYDV-GAV resistance from P. huashanica has a potential application in genetic breeding of wheat.2. A method system for amplification of BYDV-GAV full-length cDNA was constructed, based on RT-PCR amplification of cDNA segments and cDNA RACE amplification. Complete genomes of five BYDV-GAV isolates collected in different regions of Northwestern China, including GAV-AK2(KF523378), GAV-WN5(KF523379), GAV-YL4(KF523380), GAV-FF1(KF523381) and GAV-TS1(KF523382), were sequenced and bioinformatically analyzed together with all of nine BYDV-GAV genomes available in GenBank. The results showed high genome sequence identities which ranged from 97.0% to 99.7%. Among all of the seven ORFs, ORF4 showed the lowest variation and the highest genetic variation was observed in ORF1. Phylogenetic analysis based on the complete genome sequences or RdRp genes(ORF1+2) divided the 14 BYDV-GAV isolates into two groups without correlation with their geography distribution; No distinct group was observed in the phylogenetic trees based on RTP gene(ORF3/4+5), P6 gene(ORF6), P6 gene(ORF6) or P3 a gene(ORF3a). The nucleotide sequence variations in each ORF of BYDV-GAV had less effect on amino acid sequence, and the BYDV-GAV population was under a purificative selection in evolution. It is possible that the structure of population tends to be stable and the population size is expanding. There were recombination events in BYDV-GAV population. GAV-WN5 and GAV-05WN7 were both predicted to be recombinant isolates with GAV-04FX1 as major parent and GAV-38 W as minor parent.3. The full-length cDNA of BYDV-GAV Yangling isolate(GAV-YL4) was obtained by overlapping PCR and multi-segments homologous recombination method. HDV-RZ element with self-cleavage activity was fused to 3’ end of the full-length cDNA. Five infectious cDNA clone vectors were constructed, among of which pCass-GAV, pCass-GAV-RZ and pCass-HH-G-RZ were controlled by Ca MV 35 S promoter, pTCK-GAV-RZ and pRSS-GAV-RZ were controlled by maize ubiquitin promoter and rice sucrose synthase 1(RSS1) promoter, respectively. Using Agrobacterium tumefaciens strain GV3101-mediated infiltration method, genomic RNA of BYDV-GAV could be transcribed from pCass-GAV-RZ and pCass-HH-G-RZ in infiltrated leaves of Nicotiana benthamiana, and replication of genomic RNA and expression of virus protein were detectable. However, the progeny virus had no ability to move systematically. It was found that the redundant nucleotides at 3’ end of BYDV-GAV genomic RNA severely disrupted replication of virus genomic RNA or expression of virus protein, while the redundant nucleotides and cap structure at 5’ end of BYDV-GAV genomic RNA had no such effect. Using Agrobacterium tumefaciens LBA4404-mediated infiltration method, pTCK-GAV-RZ and pRSS-GAV-RZ were inoculated on seedlings of wheat and oat. A small amount of BYDV-GAV genomic RNA and their replication were detectable in inoculated leaves of wheat and oat, but the progeny virus still did not move systematically. In order to improve the infection efficiency of agrobacterium on monocot plants, different hosts(wheat, oat, rice), different agrobacterium strains(LBA4404, GV3101, AGL1, C58C1), different promoters(ubiquitin promoter, RSS1 promoter) and different inoculation methods(Agroinfiltration, vacuum infiltration, Agrobacterium acupuncture inoculation, particle bombardment) were used, although the infection efficiency was not improved. |
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