Due to its accuracy, sensitivity and high throughput, real time quantitative PCR (RT-qPCR) has been widely used in analysing gene expression. The quality of data from such analyses is affected by the quality of reference genes used. Expression stabilities for nine candidate reference genes widely used in soybean were evaluated under different stresses in this study. Our results showed that EF1A and ACT11 were the best under salinity stress, TUB4, TUA5 and EF1A were the best under drought stress, ACT11 and UKN2 were the best under dark treatment, and EF1B and UKN2 were the best under virus infection. EF1B and UKN2 were the top two genes which can be reliably used in all of the stress conditions assessed.

Salt stress caused by soil salinization, is one of the main factors that reduce soybean yield and quality. A large number of genes have been found to be involved in the regulation of salt tolerance. In this study, we characterized a soybean sodium/hydrogen exchanger gene GmNHX5 and revealed its functional mechanism involved in the salt tolerance process in soybean. GmNHX5 responded to salt stress at the transcription level in the salt stress-tolerant soybean plants, but not significantly changed in the salt-sensitive ones. GmNHX5 was located in the Golgi apparatus, and distributed in new leaves and vascular, and was induced by salt treatment. Overexpression of GmNHX5 improved the salt tolerance of hairy roots induced by soybean cotyledons, while the opposite was observed when GmNHX5 was knockout by CRISPR/Cas9. Soybean seedlings overexpressing GmNHX5 also showed an increased expression of GmSOS1, GmSKOR, and GmHKT1, higher K⁺/Na⁺ ratio, and higher viability when exposed to salt stress. Our findings provide an effective candidate gene for the cultivation of salt-tolerant germplasm resources and new clues for further understanding of the salt-tolerance mechanism in plants.

Background: Soybean (Glycine max (L.)) is one the most important oil-yielding cash crops. However, the soybean production has been seriously restricted by salinization. It is therefore crucial to identify salt tolerance-related genes and reveal molecular mechanisms underlying salt tolerance in soybean crops. A better understanding of how plants resist salt stress provides insights in improving existing soybean varieties as well as cultivating novel salt tolerant varieties. In this study, the biological function of GmNHX1, a NHX-like gene, and the molecular basis underlying GmNHX1-mediated salt stress resistance have been revealed. Results: We found that the transcription level of GmNHX1 was up-regulated under salt stress condition in soybean, reaching its peak at 24 h after salt treatment. By employing the virus-induced gene silencing technique (VIGS), we also found that soybean plants became more susceptible to salt stress after silencing GmNHX1 than wild-type and more silenced plants wilted than wild-type under salt treatment. Furthermore, Arabidopsis thaliana expressing GmNHX1 grew taller and generated more rosette leaves under salt stress condition compared to wild-type. Exogenous expression of GmNHX1 resulted in an increase of Na⁺ transportation to leaves along with a reduction of Na⁺ absorption in roots, and the consequent maintenance of a high K⁺/Na⁺ ratio under salt stress condition. GmNHX1-GFP-transformed onion bulb endothelium cells showed fluorescent pattern in which GFP fluorescence signals enriched in vacuolar membranes. Using the non-invasive micro-test technique (NMT), we found that the Na⁺ efflux rate of both wild-type and transformed plants after salt treatment were significantly higher than that of before salt treatment. Additionally, the Na⁺ efflux rate of transformed plants after salt treatment were significantly higher than that of wild-type. Meanwhile, the transcription levels of three osmotic stress-related genes, SKOR, SOS1 and AKT1 were all up-regulated in GmNHX1-expressing plants under salt stress condition. Conclusion: Vacuolar membrane-localized GmNHX1 enhances plant salt tolerance through maintaining a high K⁺/Na⁺ ratio along with inducing the expression of SKOR, SOS1 and AKT1. Our findings provide molecular insights on the roles of GmNHX1 and similar sodium/hydrogen exchangers in regulating salt tolerance.

Unc-51-like autophagy activating kinase 1 (ULK1)–autophagy-related 13 (ATG13) is the most upstream autophagy initiation complex that is phosphorylated by mammalian target-of-rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) to induce autophagy in asynchronous conditions. However, their phospho-regulation and functions in mitosis and cell cycle remain unknown. Here we show that ULK1-ATG13 complex is differentially regulated throughout the cell cycle, especially in mitosis, in which both ULK1 and ATG13 are highly phosphorylated by the key cell cycle machinery cyclin-dependent kinase 1 (CDK1)/cyclin B. Combining mass spectrometry and site-directed mutagenesis, we found that CDK1-induced ULK1-ATG13 phosphorylation promotes mitotic autophagy and cell cycle progression. Moreover, double knockout (DKO) of ULK1 and ATG13 could block cell cycle progression and significantly decrease cancer cell proliferation in cell line and mouse models. Our results not only bridge the mutual regulation between the core machinery of autophagy and mitosis but also illustrate the positive function of ULK1-ATG13 and their phosphorylation by CDK1 in mitotic autophagy regulation.

Leaf rust, caused by Puccinia triticina , is one of the most destructive fungal diseases in wheat production worldwide. The hypersensitive reaction (HR) is an important defence response against P. triticina infection. In this study, the physiological races 165 and 260 of P. triticina were combined with a line derived from the bread wheat cultivar Thatcher with the leaf rust resistance locus Lr26 to form compatible and incompatible combinations, respectively. Based on an RNA‐Seq database of the interaction systems, a new wheat cysteine‐rich receptor‐like kinase gene, TaCRK2 , is specifically induced and up‐regulated in the incompatible combination. We identified that TaCRK2 was regulated in a Ca ²⁺ ‐dependent manner. Knockdown of TaCRK2 by virus‐induced gene silencing and RNAi leads to a dramatic increase in HR area and the number of haustorial mother cells at the single infection site. In addition, urediniospores, a P. triticina‐ specific pathogenic marker in compatible combinations, were observed on leaf surfaces of silenced plants at approximately 15 days after inoculation in the incompatible combination. Moreover, transcription levels of TaPR1, TaPR2 , and TaPR5 were obviously reduced in TaCRK2 ‐silenced plants. TaCRK2 overexpression in Nicotiana benthamiana induced strong HR‐like cell death. Finally, transient expression of green fluorescent protein fused with TaCRK2 in N. benthamiana indicated that TaCRK2 localizes in the endoplasmic reticulum. Thus, TaCRK2 plays an important role in the resistance to P. triticina infection and has a positive regulation effect on the HR cell death process induced by P. triticina .

Fusarium oxysporum f. sp. lycopersici (Fol) causes vascular wilt disease in tomato. Upon colonization of the host, Fol secretes many small effector proteins into the xylem sap to facilitate infection. Besides known SIX (secreted in xylem) proteins, the identity of additional effectors that contribute to Fol pathogenicity remains largely unexplored. We performed a deep RNA-sequencing analysis of Fol race 2-infected tomato, used the sequence data to annotate a published genome assembly generated via PacBio SMRT sequencing of the Fol race 2 reference strain Fol4287, and analysed the resulting transcriptome to identify Fol effector candidates among the newly annotated genes. We examined the Fol-infection expression profiles of all 13 SIX genes present in Fol race 2 and identified 27 new candidate effector genes that were likewise significantly upregulated upon Fol infection. Using Agrobacterium-mediated transformation, we tested the ability of 22 of the new candidate effector genes to suppress or induce cell death in leaves of Nicotiana benthamiana. One effector candidate designated Fol-EC19, encoding a secreted guanyl-specific ribonuclease, was found to trigger cell death and two effector candidates designated Fol-EC14 and Fol-EC20, encoding a glucanase and a secreted trypsin, respectively, were identified that can suppress Bax-mediated cell death. Remarkably, Fol-EC14 and Fol-EC20 were also found to suppress I-2/Avr2- and I/Avr1-mediated cell death. Using the yeast secretion trap screening system, we showed that these three biologically-active effector candidates each contain a functional signal peptide for protein secretion. Our findings provide a basis for further understanding the virulence functions of Fol effectors.