Significance: Plant genomes encode hundreds of genes controlling the detection, signaling pathways, and immune responses necessary to defend against pathogens. Pathogens, in turn, continually evolve to evade these defenses. Small RNAs, such as microRNAs (miRNAs), are one mechanism used by pathogens to overcome plant defenses and facilitate plant colonization. Mounting evidence would suggest that beneficial microbes, likewise, use miRNAs to facilitate symbiosis. Here, we demonstrate that the beneficial fungus Pisolithus microcarpus encodes a miRNA that enters plant cells and stabilizes the symbiotic interaction. These results demonstrate that beneficial fungi may regulate host gene expression through the use of miRNAs and sheds light on how beneficial microbes have evolved mechanisms to colonize plant tissues.

Differential gene expression profiles often provide important clues for gene functions. While reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an important tool, the validity of the results depends heavily on the choice of proper reference genes. In this study, we employed new and published RNA-sequencing (RNA-Seq) datasets (26 sequencing libraries in total) to evaluate reference genes reported in previous soybean studies. In silico PCR showed that 13 out of 37 previously reported primer sets have multiple targets, and 4 of them have amplicons with different sizes. Using a probabilistic approach, we identified new and improved candidate reference genes. We further performed 2 validation tests (with 26 RNA samples) on 8 commonly used reference genes and 7 newly identified candidates, using RT-qPCR. In general, the new candidate reference genes exhibited more stable expression levels under the tested experimental conditions. The three newly identified candidate reference genes Bic-C2, F-box protein2, and VPS-like gave the best overall performance, together with the commonly used ELF1b. It is expected that the proposed probabilistic model could serve as an important tool to identify stable reference genes when more soybean RNA-Seq data from different growth stages and treatments are used.

To survive under abiotic stresses in the environment, plants trigger a reprogramming of gene expression, by transcriptional regulation or translational regulation, to turn on protective mechanisms. The current focus of research on how plants cope with abiotic stresses has transitioned from transcriptomic analyses to small RNA investigations. In this review, we have summarized and evaluated the current methodologies used in the identification and validation of small RNAs and their targets, in the context of plant responses to abiotic stresses.