Abstract
BackgroundArgonaute proteins are key components of RNA interference (RNAi), playing important roles in RNA-directed gene silencing. Various classes of Argonaute genes have been identified from plants and might be involved in developmental regulation. However, little is known about these genes in wheat (Triticum aestivum).ResultsIn this study, two full-length cDNAs of Argonaute were cloned from wheat, designated as TaAGO1b and TaAGO4. The cDNA of TaAGO1b is 3273 bp long and encodes 868 amino acids, with a predicted molecular weight of ~97.78 kDa and pI of 9.29. The 3157-bp TaAGO4 encodes 916 amino acids, with a molecular mass of 102.10 kDa and pI of 9.12. Genomics analysis showed that TaAGO1b and TaAGO4 contain 20 and 18 introns, respectively. Protein structural analysis demonstrated that typical PAZ and PIWI domains were found in both TaAGO1b and TaAGO4. From the highly conserved PIWI domains, we detected conserved Asp-Asp-His (DDH) motifs that function as a catalytic triad and have critical roles during the process of sequence-specific cleavage in the RNAi machinery. Structural modelling indicated that both TaAGOs can fold to a specific α/β structure. Moreover, the three aligned DDH residues are spatially close to each other at the “slicer” site of the PIWI domain. Expression analysis indicated that both genes are ubiquitously expressed in vegetative and reproductive organs, including the root, stem, leaf, anther, ovule, and seed. However, they are differentially expressed in germinating endosperm tissues. We were interested to learn that the two TaAGOs are also differentially expressed in developing wheat plants and that their expression patterns are variously affected by vernalization treatment. Further investigation revealed that they can be induced by cold accumulation during vernalization.ConclusionsTwo putative wheat Argonaute genes, TaAGO1b and TaAGO4, were cloned. Phylogenetic analysis, prediction of conserved domains and catalytic motifs, and modelling of their protein structures suggested that they encode functional Argonaute proteins. Temporal and spatial expression analyses indicated that these genes are potentially involved in developmental regulation of wheat plants.
Highlights
Argonaute proteins are key components of RNA interference (RNAi), playing important roles in RNA-directed gene silencing
Plant AGO proteins are highly conserved basic proteins with typical structural domains or amino acid motifs. These include a C-terminal PIWI (P-element induced wimpy testis) domain with an RNase-H-like fold, a central PAZ (Piwi Argonaute and Zwille) domain that binds small RNAs through the 3' end of the target RNA, and a MID domain, located between the PAZ and the PIWI domains, which anchors the 5' phosphate end of small RNAs onto Argonaute proteins [4,5]. Based on both their phylogenetic relationships and their capacity to bind to small RNAs, one can sort these proteins into three groups: I, which is the AGO-like subfamily that bind to microRNAs and small interfering RNAs; II, the PIWI-like subfamily, members of which bind to PIWIinteracting RNAs; and III, the WAGO subfamily (Worm-specific Argonautes) that bind to secondary siRNAs [5,6,7]
Reverse transcription-polymerase chain reaction (RT-PCR) amplification was conducted with the primer combination of TaAGO1-1F and -1R
Summary
Argonaute proteins are key components of RNA interference (RNAi), playing important roles in RNA-directed gene silencing. Plant AGO proteins are highly conserved basic proteins (approximately 100 kDa) with typical structural domains or amino acid motifs These include a C-terminal PIWI (P-element induced wimpy testis) domain with an RNase-H-like fold, a central PAZ (Piwi Argonaute and Zwille) domain that binds small RNAs through the 3' end of the target RNA, and a MID domain, located between the PAZ and the PIWI domains, which anchors the 5' phosphate end of small RNAs onto Argonaute proteins [4,5]. Some evidence suggests that Argonaute proteins play crucial roles in RNA-directed gene silencing, as well as being involved in the developmental regulation of plants [8,9,10]. Some of these AGO members have been characterized, most remain unexplored in plants
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