Miniature inverted repeat transposable elements (MITEs) are thought to be a driving force for genome evolution. Although numerous MITEs are found associated with genes, little is known about their function in gene regulation. Whereas the rice ubiquitin2 (rubq2) promoter in rice (Oryza sativa) line IR24 contains two nested MITEs (Kiddo and MDM1), that in line T309 has lost Kiddo, providing an opportunity to understand the role of MITEs in promoter function. No difference in endogenous rubq2 transcript levels between T309 and IR24 was evident using RT-PCR. However, promoter analysis using both transient and stably transformed calli revealed that Kiddo contributed some 20% of the total expression. Bisulfite genomic sequencing of the rubq2 promoters revealed specific DNA methylation at both symmetric and asymmetric cytosine residues on the MITE sequences, possibly induced by low levels of homologous transcripts. When methylation of the MITEs was blocked by 5-azacytidine treatment, a threefold increase in the endogenous rubq2 transcript level was detected in IR24 compared with that in T309. Together with the observed MITE methylation pattern, the detection of low levels of transcripts, but not small RNAs, corresponding to Kiddo and MDM1 suggested that RNA-dependent DNA methylation is induced by MITE transcripts. We conclude that, although Kiddo enhances transcription from the rubq2 promoter, this effect is mitigated by sequence-specific epigenetic modification.

Edited by Ruma Banerjeetrans-Aconitic acid (TAA) is an isomer of cis-aconitic acid (CAA), an intermediate of the tricarboxylic acid cycle that is synthesized by aconitase. Although TAA production has been detected in bacteria and plants for many years and is known to be a potent inhibitor of aconitase, its biosynthetic origins and the physiological relevance of its activity have remained unclear. We have serendipitously uncovered key information relevant to both of these questions. Specifically, in a search for novel nematicidal factors from Bacillus thuringiensis, a significant nematode pathogen harboring many protein virulence factors, we discovered a high yielding component that showed activity against the plant-parasitic nematode Meloidogyne incognita and surprisingly identified it as TAA. Comparison with CAA, which displayed a much weaker nematicidal effect, suggested that TAA is specifically synthesized by B. thuringiensis as a virulence factor. Analysis of mutants deficient in plasmids that were anticipated to encode virulence factors allowed us to isolate a TAA biosynthesis-related (tbr) operon consisting of two genes, tbrA and tbrB. We expressed the corresponding proteins, TbrA and TbrB, and characterized them as an aconitate isomerase and TAA transporter, respectively. Bioinformatics analysis of the TAA biosynthetic gene cluster revealed the association of the TAA genes with transposable elements relevant for horizontal gene transfer as well as a distribution across B. cereus bacteria and other B. thuringiensis strains, suggesting a general role for TAA in the interactions of B. cereus group bacteria with nematode hosts in the soil environment. This study reveals new bioactivity for TAA and the TAA biosynthetic pathway, improving our understanding of virulence factors employed by B. thuringiensis pathogenesis and providing potential implications for nematode management applications.

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Summary• RNA interference (RNAi) is of great value in plant functional genomics. However, the absence of RNAi phenotypes and the lack of uniform level of RNAi silencing has complicated gene identification. Here, the penetrance and expressivity of RNAimediated silencing of the phytoene desaturase ( PDS ) gene in Arabidopsis thaliana were examined quantitatively to provide a reference for the likely severity and distribution of silencing effects.• Arabidopsis plants were transformed with an RNAi construct targeting PDS . Transgenic plants were examined for frequency of RNAi-mediated silencing and various silencing phenotypes. mRNA depletion level and RNAi expressivity were assayed by relative reverse transcription polymerase chain reaction (RT-PCR).• High penetrance and variable expressivity of RNAi were demonstrated. An inverse correlation between PDS mRNA level and RNAi phenotype was seen. No direct relationship between copy number for the RNAi-generating transgene and phenotype was evident. Decreased RNAi penetrance in T 2 plants was observed.• It is suggested that variability in RNAi expressivity and postmeiotic decrease in RNAi penetrance constitute barriers for high throughput plant gene characterization.

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BackgroundThe apicomplexan Cryptosporidium parvum genome possesses a 25-kb intronless open reading frame (ORF) that predicts a multifunctional Type I fatty acid synthase (CpFAS1) with at least 21 enzymatic domains. Although the architecture of CpFAS1 resembles those of bacterial polyketide synthases (PKSs), this megasynthase is predicted to function as a fatty acyl elongase as our earlier studies have indicated that the N-terminal loading unit (acyl-[ACP] ligase) prefers using intermediate to long chain fatty acids as substrates, and each of the three internal elongation modules contains a complete set of enzymes to produce a saturated fatty acyl chain. Although the activities of almost all domains were confirmed using recombinant proteins, that of the C-terminal reductase domain (CpFAS1-R) was yet undetermined. In fact, there were no published studies to report the kinetic features of any reductase domains in bacterial PKSs using purified recombinant or native proteins.ResultsIn the present study, the identity of CpFAS1-R as a reductase is confirmed by in silico analysis on sequence similarity and characteristic motifs. Phylogenetic analysis based on the R-domains supports a previous notion on the bacterial origin of apicomplexan Type I FAS/PKS genes. We also developed a novel assay using fatty acyl-CoAs as substrates, and determined that CpFAS1-R could only utilize very long chain fatty acyl-CoAs as substrates (i.e., with activity on C26 > C24 > C22 > C20, but no activity on C18 and C16). It was capable of using both NADPH and NADH as electron donors, but prefers NADPH to NADH. The activity of CpFAS1-R displayed allosteric kinetics towards C26 hexacosanoyl CoA as a substrate (h = 2.0; Vmax = 32.8 nmol min-1 mg-1 protein; and K50 = 0.91 mM).ConclusionsWe have confirmed the activity of CpFAS1-R by directly assaying its substrate preference and kinetic parameters, which is for the first time for a Type I FAS, PKS or non-ribosomal peptide synthase (NRPS) reductase domain. The restricted substrate preference towards very long chain fatty acyl thioesters may be an important feature for this megasynthase to avoid the release of product(s) with undesired lengths.

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