ABSTRACT
The functions of RNA silencing are repression of endogenous gene expression and antiviral defense in plants and animals. Cucumber mosaic virus 2b (CMV2b) is a suppressor of RNA silencing in higher plants. In the present study, we evaluated the RNA silencing suppressor activity of CMV2b in Chlamydomonas reinhardtii. Before transformation, we modified CMV2b codons to increase the GC content for optimal expression in C. reinhardtii. Inhibition of Maa7 silencing was detected in CMV2b-expressing Maa7-IR44 strains, indicating that CMV2b suppressed siRNA pathways in C. reinhardtii as in higher plants. In addition, mRNA expression targeted for cleavage by miRNA was significantly higher in CMV2b-expressing strains, but increased accumulation of miRNA was not detected. These results indicate that the suppression of miRNA pathways is mediated by CMV2b in C. reinhardtii. Interestingly, expression of both Argonaute 1 (AGO1) and Dicer-like 1 (DCL1), regulated by a bidirectional promoter, was reduced in CMV2b-expressing strains, suggesting that CMV2b may affect transcription factors involved in RNA silencing pathways. Furthermore, reduction of AGO2 and AGO3 expression was detected in CMV2b-expressing strains. Taken together, our results demonstrate that CMV2b may suppress both siRNA and miRNA pathways, and also impair AGOs and DCL1 expression in C. reinhardtii.
Subject(s)
Chlamydomonas reinhardtii/genetics , RNA Interference , Viral Proteins/metabolism , Algal Proteins/metabolism , Amino Acid Sequence , Base Sequence , Cucumovirus/genetics , Cucumovirus/metabolism , Gene Expression Regulation , MicroRNAs/genetics , Molecular Sequence Data , RNA, Algal/genetics , RNA, Messenger/genetics , Viral Proteins/geneticsABSTRACT
During batch cultivation of Agrobacterium sp. ATCC 31750, proteome analysis in response to a pH downshift from 7.0 to 5.5 was carried out using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. When the pH of the exponentially growing Agrobacterium sp. culture was downshifted to pH 5.5, the synthesis level of 27 intracellular proteins showed significant changes in level over a prolonged period of time compared with the batch culture controlled at pH 7.0. In particular, the intracellular protein level of the beta-1,3-glucan synthase catalytic subunit, UTP-glucose-1-phosphate uridylyltransferase, and phosphoglucomutase, which are key metabolic enzymes in the curdlan biosynthesis pathway, were more than 10-, 3- and 17-times higher in the low pH culture. On the other hand, the level of orotidine5-phosphate decarboxylase (conversion of OMP to UMP) was significantly up-regulated after pH downshift. The accumulation of UMP may direct the metabolic flow towards the biosynthetic route of UTP, which is a key metabolic precursor for UDP-glucose. Therefore, it is possible that increase of cellular metabolic enzymes during pH downshift culture can enhance the metabolic flux of the biosynthesis of key precursor, such as UTP- and UDP-glucose, resulting in an increase in curdlan biosynthesis.
