Optimization and Application of a Quantitative Polymerase Chain Reaction Assay to Detect Diaporthe Species in Soybean Plant Tissue.

Diaporthe caulivora and D. longicolla are the causal agents of stem canker of soybean (Glycine max L.). Accurate identification of stem canker pathogens upon isolation from infected soybean plants is difficult and unreliable based on morphology. In this study, two TaqMan probe-based quantitative polymerase chain reaction (qPCR) assays were optimized for detection of D. caulivora and D. longicolla in soybean plants. The assays used previously reported D. caulivora-specific (DPC-3) and D. longicolla-specific (PL-3) probe/primer sets. The sensitivity limit of the two assays was determined to be over a range of 100 pg to 10 fg of pure D. caulivora and D. longicolla genomic DNA. The qPCR assays were validated with plant samples collected from commercial soybean fields. The PL-3 set detected D. longicolla in soybean plants collected from the fields (quantification cycle value <35), which was confirmed by isolation on potato dextrose agar (PDA). D. caulivora was detected only in low levels (quantification cycle value <40) by DPC-3 set in a few of the symptomatic field samples, although the pathogen was not isolated on PDA. The qPCR assays were also useful in quantitatively phenotyping soybean plants for resistance to D. caulivora and D. longicolla under greenhouse conditions.

microRNA160 dictates stage-specific auxin and cytokinin sensitivities and directs soybean nodule development.

Legume nodules result from coordinated interactions between the plant and nitrogen-fixing rhizobia. The phytohormone cytokinin promotes nodule formation, and recent findings suggest that the phytohormone auxin inhibits nodule formation. Here we show that microRNA160 (miR160) is a key signaling element that determines the auxin/cytokinin balance during nodule development in soybean (Glycine max). miR160 appears to promote auxin activity by suppressing the levels of the ARF10/16/17 family of repressor ARF transcription factors. Using quantitative PCR assays and a fluorescence miRNA sensor, we show that miR160 levels are relatively low early during nodule formation and high in mature nodules. We had previously shown that ectopic expression of miR160 in soybean roots led to a severe reduction in nodule formation, coupled with enhanced sensitivity to auxin and reduced sensitivity to cytokinin. Here we show that exogenous cytokinin restores nodule formation in miR160 over-expressing roots. Therefore, low miR160 levels early during nodule development favor cytokinin activity required for nodule formation. Suppression of miR160 levels using a short tandem target mimic (STTM160) resulted in reduced sensitivity to auxin and enhanced sensitivity to cytokinin. In contrast to miR160 over-expressing roots, STTM160 roots had increased nodule formation, but nodule maturation was significantly delayed. Exogenous auxin partially restored proper nodule formation and maturation in STTM160 roots, suggesting that high miR160 activity later during nodule development favors auxin activity and promotes nodule maturation. Therefore, miR160 dictates developmental stage-specific sensitivities to auxin and cytokinin to direct proper nodule formation and maturation in soybean.

Ectopic expression of miR160 results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of symbiotic nodule development in soybean.

Symbiotic root nodules in leguminous plants result from interaction between the plant and nitrogen-fixing rhizobia bacteria. There are two major types of legume nodules, determinate and indeterminate. Determinate nodules do not have a persistent meristem, while indeterminate nodules have a persistent meristem. Auxin is thought to play a role in the development of both these types of nodules. However, inhibition of rootward auxin transport at the site of nodule initiation is crucial for the development of indeterminate nodules but not determinate nodules. Using the synthetic auxin-responsive DR5 promoter in soybean (Glycine max), we show that there is relatively low auxin activity during determinate nodule initiation and that it is restricted to the nodule periphery subsequently during development. To examine if and what role auxin plays in determinate nodule development, we generated soybean composite plants with altered sensitivity to auxin. We overexpressed microRNA393 to silence the auxin receptor gene family, and these roots were hyposensitive to auxin. These roots nodulated normally, suggesting that only minimal/reduced auxin signaling is required for determinate nodule development. We overexpressed microRNA160 to silence a set of repressor auxin response factor transcription factors, and these roots were hypersensitive to auxin. These roots were not impaired in epidermal responses to rhizobia but had significantly reduced nodule primordium formation, suggesting that auxin hypersensitivity inhibits nodule development. These roots were also hyposensitive to cytokinin and had attenuated expression of key nodulation-associated transcription factors known to be regulated by cytokinin. We propose a regulatory feedback loop involving auxin and cytokinin during nodulation.

Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs.

We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays.

Effects of triclosan on seed germination and seedling development of three wetland plants: Sesbania herbacea, Eclipta prostrata, and Bidens frondosa.

Three wetland macrophytes, Sesbania herbacea, Bidens frondosa, and Eclipta prostrata, were exposed (0.4-1,000-ppb nominal concentrations) to the antimicrobial triclosan for 28 d in a flow-through system. Sesbania herbacea had decreased seed germination at the 100-ppb exposure level at days 7, 14, and 21, and B. frondosa germination was reduced at the 1,000-ppb exposure level at day 7. Eclipta prostrata germination was unaffected. Seedling effects monitored were total fresh weight, shoot and root fresh weights, root length, and root surface area. Root metrics were most affected by exposure. Total root length was diminished at all exposure levels in S. herbacea and B. frondosa and at the 10-ppb and higher concentrations for E. prostrata. Root surface area decreased at all exposure levels in B. frondosa and at the 10-ppb level and above in S. herbacea and E. prostrata. Root and shoot bioconcentration factors (BCFs) were estimated for S. herbacea and B. frondosa. While BCFs were low in shoots of both species and roots of S. herbacea (<10), they were elevated in B. frondosa roots (53-101). Methyl-triclosan was formed in the system and accumulated in shoot and root tissues of S. herbacea to concentrations that exceeded those of the parent compound. However, methyl-triclosan was nontoxic in an Arabidopsis thaliana enoyl-acyl carrier protein reductase (the putative enzymatic target of triclosan) assay and did not appear to contribute to the effects of exposure. Two of the three plant species assessed exhibited reduced root systems at environmentally relevant concentrations, raising the concern that wetland plant performance could be compromised in constructed wetlands receiving wastewater treatment plant discharges.