ABSTRACT
During their infection in plants, viruses can form double stranded (ds) RNA structures. These dsRNAs can be recognized by plants as "aberrant" signals and short interfering RNA (siRNA) molecules of 19-25 nt will be produced with sequences derived from the viral source. Knowledge about antiviral siRNA profiles including siRNA size, distribution, polarity, etc. provides valuable insights to plant-virus interactions. In this chapter, we describe a simple method for cloning siRNA from virus-infected plants. This protocol includes isolation of small RNAs, their ligation to a pair of 5' and 3' adapters, RT-PCR/PCR amplification, and subsequent concatamerization before pGEM-T cloning and sequencing. Concatamers containing as many as 15 small RNA inserts can be produced. This protocol has successfully been applied to leaf materials of monocots and dicots infected with poty-, carmo-, and sobemo-viruses.
Subject(s)
Plant Diseases/virology , RNA, Plant/genetics , RNA, Small Interfering/genetics , RNA, Viral/genetics , Cloning, Molecular/methods , Plants/virology , RNA Interference , RNA, Double-Stranded/genetics , RNA, Plant/isolation & purification , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
The Brassica rapa line RLR22 was resistant to eight diverse turnip mosaic virus (TuMV) isolates. A B. rapa genetic map based on 213 marker loci segregating in 120 first back-cross (B(1)) individuals was established and aligned with the B. rapa genome reference map using some of the RFLP probes. B(1) individuals were self-pollinated to produce B(1)S(1) families. The existence of two loci controlling resistance to TuMV isolate CDN 1 was established from contrasting patterns of segregation for resistance and susceptibility in the B(1)S(1) families. The first gene, recessive TuMV resistance 01 (retr01), had a recessive allele for resistance, was located on the upper portion of chromosome R4 and was epistatic to the second gene. The second gene, Conditional TuMV resistance 01 (ConTR01), possessed a dominant allele for resistance and was located on the upper portion of chromosome R8. These genes also controlled resistance to TuMV isolate CZE 1 and might be sufficient to explain the broad-spectrum resistance of RLR22. The dominant resistance gene, ConTR01, was coincident with one of the three eukaryotic initiation factor 4E (eIF4E) loci of B. rapa and possibly one of the loci of eIF(iso)4E. The recessive resistance gene retr01 was apparently coincident with one of the three loci of eIF(iso)4E in the A genome of Brassica napus and therefore, by inference, in the B. rapa genome. This suggested a mode of action for the resistance that is based on denying the viral RNA access to the translation initiation complex of the plant host. The gene retr01 is the first reported example of a recessive resistance gene mapped in a Brassica species.
Subject(s)
Brassica rapa/virology , Immunity, Innate/genetics , Plant Diseases/virology , Potyvirus/growth & development , Chromosome Mapping , Chromosomes, Plant , Eukaryotic Initiation Factor-4E/genetics , Genes, Dominant , Genes, Plant , Genes, Recessive , Protein BiosynthesisABSTRACT
RNA silencing is a plant defense mechanism in which virus infected plants produce short interfering RNAs (siRNAs) derived from viral RNA, that attack the virus at the post-transcriptional level. In a previous study on Cymbidium ringspot tombusvirus (CymRSV) infection in Nicotiana benthamiana, siRNAs (determined by cloning and sequencing) predominantly originated from the sense (+) strand of the viral RNA, suggesting that the majority of siRNAs are produced through the direct cleavage of the virus single strand (ss) RNA by the plant Dicer-like enzyme. To test whether this asymmetry in strand polarity is a generic rule for all plant viruses, siRNAs from Brassica juncea, either singly infected by Turnip mosaic potyvirus (TuMV, the family Potyviridae), or doubly infected with TuMV and Turnip crinkle carmovirus (TCV, the family Tombusviridae) were investigated. A simplified siRNA cloning method was developed, using a single ligation reaction to attach both 5' and 3' adapters to the target short RNAs followed by one-step RT-PCR amplification. In the TCV infection, as for the CymRSV infection, siRNAs were produced predominantly (97.6%) from the +ss RNA. However, for TuMV infections, siRNAs were derived from both strands (+/-, 58.1-41.9%), indicating the presence of alternative siRNA production mechanisms.
Subject(s)
Carmovirus/genetics , Cloning, Molecular/methods , Mustard Plant/virology , Potyvirus/genetics , RNA, Small Interfering/genetics , RNA, Viral/genetics , Electrophoresis, Polyacrylamide Gel , RNA/genetics , RNA/isolation & purification , RNA, Small Interfering/isolation & purification , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The wild apple (Malus sieversii) is a large-fruited species from Central Asia, which is used as a source of scab resistance in cultivar breeding. Phytopathological tests with races of Venturia inaequalis were performed to differentiate scab-resistance genes in Malus as well as an avirulence gene in the pathogen. A novel gene-for-gene interaction between V. inaequalis and Malus was identified. The locus of the scab-resistance gene Vh8 is linked with, or possibly allelic to, that of the Vh2 gene in Malus pumila Russian apple R12740-7A, at the lower end of linkage group 2 of Malus. Race 8 isolate NZ188B.2 is compatible with Vh8, suggesting the loss or modification of the complementary AvrVh8 gene, while isolate 1639 overcomes both Vh2 and Vh8, but is incompatible with at least one other gene not detected by any of the other race isolates tested. Our research is the first to differentiate scab-resistance genes in a putative gene cluster in apple with the aid of races of V. inaequalis.
