A 14-3-3 protein positively regulates rice salt tolerance by stabilizing phospholipase C1.

The phosphatidylinositol-specific phospholipase Cs (PI-PLCs) catalyze the hydrolysis of phosphatidylinositols, which play crucial roles in signaling transduction during plant development and stress response. However, the regulation of PI-PLC is still poorly understood. A previous study showed that a rice PI-PLC, OsPLC1, was essential to rice salt tolerance. Here, we identified a 14-3-3 protein, OsGF14b, as an interaction partner of OsPLC1. Similar to OsPLC1, OsGF14b also positively regulates rice salt tolerance, and their interaction can be promoted by NaCl stress. OsGF14b also positively regulated the hydrolysis activity of OsPLC1, and is essential to NaCl-induced activation of rice PI-PLCs. We further discovered that OsPLC1 was degraded via ubiquitin-proteasome pathway, and OsGF14b could inhibit the ubiquitination of OsPLC1 to protect OsPLC1 from degradation. Under salt stress, the OsPLC1 protein level in osgf14b was lower than the corresponding value of WT, whereas overexpression of OsGF14b results in a significant increase of OsPLC1 stability. Taken together, we propose that OsGF14b can interact with OsPLC1 and promote its activity and stability, thereby improving rice salt tolerance. This study provides novel insights into the important roles of 14-3-3 proteins in regulating protein stability and function in response to salt stress.

The transcription factor OsMYBc and an E3 ligase regulate expression of a K+ transporter during salt stress.

Sodium (Na+) and potassium (K+) homeostasis is essential for plant survival in saline soils. A member of the High-Affinity K+ Transporter (HKT) family in rice (Oryza sativa), OsHKT1;1, is a vital regulator of Na+ exclusion from shoots and is bound by a MYB transcription factor (OsMYBc). Here, we generated transgenic rice lines in the oshkt1;1 mutant background for genetic complementation using genomic OsHKT1;1 containing a native (Com) or mutated (mCom) promoter that cannot be bound by OsMYBc. In contrast to wild-type (WT) or Com lines, the mCom lines were not able to recover the salt-sensitive phenotype of oshkt1;1. The OsMYBc-overexpressing plants were more tolerant to salt stress than WT plants. A yeast two-hybrid screen using the OsMYBc N-terminus as bait identified a rice MYBc stress-related RING finger protein (OsMSRFP). OsMSRFP is an active E3 ligase that ubiquitinated OsMYBc in vitro and mediated 26S proteasome-mediated degradation of OsMYBc under semi-in vitro and in vivo conditions. OsMSRFP attenuated OsMYBc-mediated OsHKT1;1 expression, and knockout of OsMSRFP led to rice salt tolerance. These findings uncover a regulatory mechanism of salt response that fine-tunes OsHKT1;1 transcription by ubiquitination of OsMYBc.

An endoplasmic reticulum-localized cytochrome b5 regulates high-affinity K+ transport in response to salt stress in rice.

Potassium (K+) is an essential element for growth and development in both animals and plants, while high levels of environmental sodium (Na+) represent a threat to most plants. The uptake of K+ from high-saline environments is an essential mechanism to maintain intracellular K+/Na+ homeostasis, which can help reduce toxicity caused by Na+ accumulation, thereby improving the salt tolerance of plants. However, the mechanisms and regulation of K+-uptake during salt stress remain poorly understood. In this study, we identified an endoplasmic reticulum-localized cytochrome b5 (OsCYB5-2) that interacted with a high-affinity K+ transporter (OsHAK21) at the plasma membrane. The association of OsCYB5-2 with the OsHAK21 transporter caused an increase in transporter activity by enhancing the apparent affinity for K+-binding but not Na+-binding. Heme binding to OsCYB5-2 was essential for the regulation of OsHAK21. High salinity directly triggered the OsHAK21-OsCYB5-2 interaction, promoting OsHAK21-mediated K+-uptake and restricting Na+ entry into cells; this maintained intracellular K+/Na+ homeostasis in rice cells. Finally, overexpression of OsCYB5-2 increased OsHAK21-mediated K+ transport and improved salt tolerance in rice seedlings. This study revealed a posttranslational regulatory mechanism for HAK transporter activity mediated by a cytochrome b5 and highlighted the coordinated action of two proteins to perceive Na+ in response to salt stress.

Phosphatidic acid directly binds with rice potassium channel OsAKT2 to inhibit its activity.

The plant Shaker K+ channel AtAKT2 has been identified as a weakly rectifying channel that can stabilize membrane potentials to promote photoassimilate phloem loading and translocation. Thus, studies on functional characterization and regulatory mechanisms of AtAKT2-like channels in crops are highly important for improving crop production. Here, we identified the rice OsAKT2 as the ortholog of Arabidopsis AtAKT2, which is primarily expressed in the shoot phloem and localized at the plasma membrane. Using an electrophysiological assay, we found that OsAKT2 operated as a weakly rectifying K+ channel, preventing H+ /sucrose-symport-induced membrane depolarization. Three critical amino acid residues (K193, N206, and S326) are essential to the phosphorylation-mediated gating change of OsAKT2, consistent with the roles of the corresponding sites in AtAKT2. Disruption of OsAKT2 results in delayed growth of rice seedlings under short-day conditions. Interestingly, the lipid second messenger phosphatidic acid (PA) inhibits OsAKT2-mediated currents (both instantaneous and time-dependent components). Lipid dot-blot assay and liposome-protein binding analysis revealed that PA directly bound with two adjacent arginine residues in the ANK domain of OsAKT2, which is essential to PA-mediated inhibition of OsAKT2. Electrophysiological and phenotypic analyses also showed the PA-mediated inhibition of AtAKT2 and the negative correlation between intrinsic PA level and Arabidopsis growth, suggesting that PA may inhibit AKT2 function to affect plant growth and development. Our results functionally characterize the Shaker K+ channel OsAKT2 and reveal a direct link between phospholipid signaling and plant K+ channel modulation.

A new point mutation in ß2-tubulin confers resistance to carbendazim in Fusarium asiaticum.

Resistance to benzimidazole fungicides in many phytopathogenic fungi is caused by specific point mutations in the ß-tubulin gene (ß-tubulin). However, the mutated locus and genotype of ß-tubulin differ among phytopathogenic fungi. To validate the point mutation in Fusarium asiaticum ß2-tubulin that confers resistance to carbendazim and to analyze the molecular interaction between carbendazim and F. asiaticum ß2-tubulin. In this study, a new point mutation (GAG→GCG, E198A) at codon 198 of ß2-tubulin in a wild-type F. asiaticum strain was constructed by site-directed mutagenesis followed by a split marker strategy. The site-directed mutants were verified and exhibited a high level of resistance to carbendazim. In the absence of fungicide treatment, the biological characteristics did not differ between the site-directed mutants and the wild-type strain. Molecular docking between carbendazim and ß2-tubulin was carried out using the Surflex-Dock program in Sybyl X-2.0 version and the results indicated that the E198A mutation altered the configuration of ß2-tubulin, resulting in the change of the bonding sites and docking scores. We concluded that the point mutation of F. asiaticum ß2-tubulin conferring carbendazim resistance may not always be the bonding site for carbendazim.

Development of a rapid and high-throughput molecular method for detecting the F200Y mutant genotype in benzimidazole-resistant isolates of Fusarium asiaticum.

BACKGROUND: The point mutation at codon 200 (TTC→TAC, F200Y) of the ß2 -tubulin gene confers resistance to benzimidazole fungicide in Fusarium asiaticum. These isolates with this mutation have been detected mainly by determining the minimum inhibitory concentration (MIC) of fungicides, which is always time consuming, tedious and inefficient. RESULTS: A visual, rapid and efficient method with high specificity was developed, based on loop-mediated isothermal amplification (LAMP). Six sets of LAMP primers were designed, and one set was optimised specifically to distinguish the F200Y mutant genotype. With the optimal LAMP primers, concentrations of LAMP components were optimised. The optimal reaction conditions were 57-64 °C for 75 min. The feasibility of the LAMP assay for detection of the F200Y mutant genotype of F. asiaticum was demonstrated by assaying diseased wheat spikelets that were artificially inoculated in the field. CONCLUSION: The new LAMP assay had good specificity, sensitivity, stability and repeatability. It will be useful for assessing the risk of F. asiaticum populations with carbendazim resistance developing in the field, and will also provide important reference data for integrated control of Fusarium head blight caused by F. asiaticum. © 2016 Society of Chemical Industry.