The interaction of two Puccinia striiformis f. sp. tritici effectors modulates high-temperature seedling-plant resistance in wheat.

Wheat cultivar Xiaoyan 6 (XY6) has high-temperature seedling-plant (HTSP) resistance to Puccinia striiformis f. sp. tritici (Pst). However, the molecular mechanism of Pst effectors involved in HTSP resistance remains unclear. In this study, we determined the interaction between two Pst effectors, PstCEP1 and PSTG_11208, through yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and pull-down assays. Transient overexpression of PSTG_11208 enhanced HTSP resistance in different temperature treatments. The interaction between PstCEP1 and PSTG_11208 inhibited the resistance enhancement by PSTG_11208. Furthermore, the wheat apoplastic thaumatin-like protein 1 (TaTLP1) appeared to recognize Pst invasion by interacting with PSTG_11208 and initiate the downstream defence response by the pathogenesis-related protein TaPR1. Silencing of TaTLP1 and TaPR1 separately or simultaneously reduced HTSP resistance to Pst in XY6. Moreover, we found that PstCEP1 targeted wheat ferredoxin 1 (TaFd1), a homologous protein of rice OsFd1. Silencing of TaFd1 affected the stability of photosynthesis in wheat plants, resulting in chlorosis on the leaves and reducing HTSP resistance. Our findings revealed the synergistic mechanism of effector proteins in the process of pathogen infection.

Genomic analysis, trajectory tracking, and field surveys reveal sources and long-distance dispersal routes of wheat stripe rust pathogen in China.

Identifying sources of phytopathogen inoculum and determining their contributions to disease outbreaks are essential for predicting disease development and establishing control strategies. Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, is an airborne fungal pathogen with rapid virulence variation that threatens wheat production through its long-distance migration. Because of wide variation in geographic features, climatic conditions, and wheat production systems, Pst sources and related dispersal routes in China are largely unclear. In the present study, we performed genomic analyses of 154 Pst isolates from all major wheat-growing regions in China to determine Pst population structure and diversity. Through trajectory tracking, historical migration studies, genetic introgression analyses, and field surveys, we investigated Pst sources and their contributions to wheat stripe rust epidemics. We identified Longnan, the Himalayan region, and the Guizhou Plateau, which contain the highest population genetic diversities, as the Pst sources in China. Pst from Longnan disseminates mainly to eastern Liupan Mountain, the Sichuan Basin, and eastern Qinghai; that from the Himalayan region spreads mainly to the Sichuan Basin and eastern Qinghai; and that from the Guizhou Plateau migrates mainly to the Sichuan Basin and the Central Plain. These findings improve our current understanding of wheat stripe rust epidemics in China and emphasize the need for managing stripe rust on a national scale.

A Puccinia striiformis f. sp. tritici effector inhibits high-temperature seedling-plant resistance in wheat.

Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1)-induced protein kinase (RIPK) in Arabidopsis belongs to the receptor-like cytoplasmic kinase (RLCK) family and plays a vital role in immunity. However, the role of RLCKs in the high-temperature seedling-plant (HTSP) resistance of wheat (Triticum aestivum) to Puccinia striiformis f. sp. tritici (Pst), the stripe rust pathogen, remains unclear. Here, we identified a homologous gene of RIPK in wheat, namely TaRIPK. Expression of TaRIPK was induced by Pst inoculation and high temperatures. Silencing of TaRIPK reduced the expression level of TaRPM1, resulting in weaker HTSP resistance. Moreover, TaRIPK interacts with and phosphorylates papain-like cysteine protease 1 (TaPLCP1). Meanwhile, we found that the Pst-secreted protein PSTG_01766 targets TaPLCP1. Transient expression of PSTG_01766 inhibited basal immunity in tobacco (Nicotiana benthamiana) and wheat. The role of PSTG_01766 as an effector involved in HTSP resistance was further supported by host-induced gene silencing and bacterial type three secretion system-mediated delivery into wheat. PSTG_01766 inhibited the TaRIPK-induced phosphorylation of TaPLCP1. Furthermore, PSTG_01766 has the potential to influence the subcellular localization of TaPLCP1. Overall, we suggest that the TaRIPK-TaPLCP1-TaRPM1 module fits the guard model for disease resistance, participating in HTSP resistance. PSTG_01766 decreases HTSP resistance via targeting TaPLCP1. Guarded by wheat and attacked by Pst, TaPLCP1 may serve as a central hub of the defense response. Our findings improve the understanding of the molecular mechanism of wheat HTSP resistance, which may be an important strategy for controlling stripe rust in the face of global warming.

The RLK protein TaCRK10 activates wheat high-temperature seedling-plant resistance to stripe rust through interacting with TaH2A.1.

Plants sense various pathogens and activate immunity responses through receptor-like kinases (RLKs). Cysteine-rich receptor-like kinases (CRKs) are involved in massive transduction pathways upon perception of a pathogen. However, the roles of CRKs in response to stripe rust are unclear. In the present study, we identified a CRK gene (designated TaCRK10) from wheat variety Xiaoyan 6 (XY6) that harbors high-temperature seedling-plant (HTSP) resistance to stripe rust caused by fungal pathogen Puccinia striiformis f. sp. tritici (Pst). The expression level of TaCRK10 was induced by Pst inoculation and high temperature treatment. Knockdown of TaCRK10 by virus-induced gene silencing resulted in attenuated wheat HTSP resistance to Pst, whereas there is no effect on Pst development and host responses under normal temperatures. Notably, overexpression of TaCRK10 in susceptible variety Fielder provided resistance only under normal temperatures at 14 days with reactive oxygen species accumulation and defense-related gene expression of the salicylic acid pathway. Moreover, TaCRK10 physically interacted with and phosphorylated a histone variant TaH2A.1, which belongs to the H2A.W group. Silencing of TaH2A.1 suppressed wheat resistance to Pst, indicating that TaH2A.1 plays a positive role in wheat resistance to Pst. Thus, TaCRK10 serves as an important sensor of Pst infection and high temperatures, and it activates wheat resistance to Pst through regulating nuclear processes. This knowledge helps elucidate the molecular mechanism of wheat HTSP resistance to Pst and promotes efforts in developing wheat varieties with resistance to stripe rust.

NBS-LRR Gene TaRPS2 is Positively Associated with the High-Temperature Seedling Plant Resistance of Wheat Against Puccinia striiformis f. sp. tritici.

Xiaoyan6 (XY6) is a wheat (Triticum aestivum) cultivar possessing nonrace-specific high-temperature seedling plant (HTSP) resistance against stripe rust, caused by Puccinia striiformis f. sp. tritici. Previously, we identified one particular gene, TaRPS2, for its involvement in the HTSP resistance. To elucidate the role of TaRPS2 in the HTSP resistance, we cloned the full length of TaRPS2 from XY6. The transcriptional expression of TaRPS2 was rapidly upregulated (19.11-fold) under the normal-high-normal temperature treatment that induces the HTSP resistance. The expression level of TaRPS2 in leaves was higher than that in the stems and roots. Quantification of the endogenous hormones in wheat leaves after P. striiformis f. sp. tritici inoculation showed that 1-aminocyclopropane-1-carboxylic acid, salicylic acid (SA), and jasmonic acid were involved in the HTSP resistance. In addition, detection of hydrogen peroxide (H2O2) accumulation indicated that reactive oxygen species burst was also associated with the HTSP resistance. Two hours after exogenous H2O2 treatment or 0.5 h after SA treatment, the expression level of TaRPS2 was increased by 2.66 and 2.35 times, respectively. The subcellular localization of enhanced green fluorescent protein-TaRPS2 fusion protein was in the nuclei and plasma membranes. Virus-induced gene silencing of TaRPS2 reduced the level of HTSP resistance in XY6. Compared with the nonsilenced leaves, the TaRPS2-silenced leaves had the reduction of necrotic cells but a greater number of uredinia. These results indicated that TaRPS2 positively regulates the HTSP resistance of XY6 against P. striiformis f. sp. tritici and is related to the SA and H2O2 signaling pathways.

TaXa21, a Leucine-Rich Repeat Receptor-Like Kinase Gene Associated with TaWRKY76 and TaWRKY62, Plays Positive Roles in Wheat High-Temperature Seedling Plant Resistance to Puccinia striiformis f. sp. tritici.

Puccinia striiformis f. sp. tritici causes wheat stripe rust, one of most important diseases of wheat worldwide. High-temperature seedling plant (HTSP) resistance of wheat to P. striiformis f. sp. tritici is one specific type of host resistance, induced by high temperature (HT). Receptor-like kinases (RLKs) play key roles in regulating plant development and signaling networks, but there have been no reports on possible roles played by RLKs in wheat HTSP to P. striiformis f. sp. tritici. In the present study, a leucine rich repeat (LRR)-RLK gene, TaXa21, with a high homology with rice bacterial blight resistance gene Xa21, was cloned from wheat cultivar Xiaoyan 6 (XY 6). TaXa21 expression was up-regulated by the exposure to HT (20°C) for 24 h at 8 days postinoculation with P. striiformis f. sp. tritici and was induced by ethylene (ET) and hydrogen peroxide (H2O2). Knocking down TaXa21 using virus-induced gene silencing reduced HTSP resistance to P. striiformis f. sp. tritici compared with the control plants. In addition, the expression level of TaCAT in the H2O2 pathway was induced and TaACO in the ET signal pathway was reduced in the HT-treated TaXa21-silenced plants. Transient expression of TaXa21 in tobacco leaves confirmed its subcellular localization in plasma membrane, consistent with the prediction from bioinformatics analysis. The transmembrane and kinase domain of TaXa21 can interact with TaWRKY76 in the nucleus and cell membrane, which is different from the localization of Xa21 in rice. The interaction between TaWRKY76 and TaWRKY62 (positively involved in the HTSP resistance of XY 6) were observed. Together, these results indicated that TaXa21 is a RLK associated with TaWRKY76 and TaWRKY62 and functions as a positive regulator of wheat HTSP resistance to P. striiformis f. sp. tritici. Furthermore, the host defense is mediated by the H2O2 and ET signal pathways.

TaRPM1 Positively Regulates Wheat High-Temperature Seedling-Plant Resistance to Puccinia striiformis f. sp. tritici.

RPM1 is a CC-NBS-LRR protein that was first shown to be required for resistance to Pseudomonas syringae pv. maculicola in Arabidopsis thaliana. Our previous study showed that TaRPM1 gene in wheat was upregulated about six times following infection by Puccinia striiformis f. sp. tritici (Pst) under high temperature, compared with normal temperature. To study the function of TaRPM1 in wheat high-temperature seedling-plant (HTSP) resistance to Pst, the full length of TaRPM1 was cloned, with three copies each located on chromosomes 1A, 1B, and 1D. Transient expression of the TaRPM1-GFP fusion protein in Nicotiana benthamiana indicated that TaRPM1 localizes in the cytoplasm and nucleus. Profiling TaRPM1 expression indicated that TaRPM1 transcription was rapidly upregulated upon Pst inoculation under high temperature. In addition, TaRPM1 was induced by exogenous salicylic acid hormone application. Silencing TaRPM1 in wheat cultivar Xiaoyan 6 (XY 6) resulted in reduced HTSP resistance to Pst in terms of reduced number of necrotic cells and increased uredinial length, whereas no obvious phenotypic changes were observed in TaRPM1-silenced leaves under normal temperature. Related defense genes TaPR1 and TaPR2 were downregulated in TaRPM1-silenced plants under high temperature. We conclude that TaRPM1 is involved in HTSP resistance to Pst in XY 6.

The wheat WRKY transcription factors TaWRKY49 and TaWRKY62 confer differential high-temperature seedling-plant resistance to Puccinia striiformis f. sp. tritici.

WRKY transcription factors (TFs) play crucial roles in plant resistance responses to pathogens. Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat (Triticum aestivum) worldwide. In this study, the two WRKY genes TaWRKY49 and TaWRKY62 were originally identified in association with high-temperature seedling-plant resistance to Pst (HTSP) resistance in wheat cultivar Xiaoyan 6 by RNA-seq. Interestingly, the expression levels of TaWRKY49 and TaWRKY62 were down- and up-regulated, respectively, during HTSP resistance in response to Pst. Silencing of TaWRKY49 enhanced whereas silencing TaWRKY62 reduced HTSP resistance. The enhanced resistance observed on leaves following the silencing of TaWRKY49 was coupled with increased expression of salicylic acid (SA)- and jasmonic acid (JA)-responsive genes TaPR1.1 and TaAOS, as well as reactive oxygen species (ROS)-associated genes TaCAT and TaPOD; whereas the ethylene (ET)-responsive gene TaPIE1 was suppressed. The decreased resistance observed on leaves following TaWRKY62 silencing was associated with increased expression of TaPR1.1 and TaPOD, and suppression of TaAOS and TaPIE1. Furthermore, SA, ET, MeJA (methyl jasmonate), hydrogen peroxide (H2O2) and abscisic acid (ABA) treatments increased TaWRKY62 expression. On the other hand, MeJA did not affect the expression of TaWRKY49, and H2O2 reduced TaWRKY49 expression. In conclusion, TaWRKY49 negatively regulates while TaWRKY62 positively regulates wheat HTSP resistance to Pst by differential regulation of SA-, JA-, ET and ROS-mediated signaling.

[Closed reduction and percutaneous double K-wires internal fixation for the treatment of multisegmental fracture of humeral shaft].

OBJECTIVE: To observe the clinical effects of closed reduction and percutaneous double K-wires internal fixation in treating multisegmental fracture of humeral shaft. METHODS: From January 2009 to April 2015, 27 patients with multisegmental fracture of humeral shaft were treated with closed reduction and percutaneous double K-wires internal fixation, including 10 males and 17 females, ranging in age from 26 to 81 years with an average of 52 years;the disease course ranged from 2 hours to 6 days with an average of 1.5 days. Operative time, intraoperative blood loss, hospital stay, fracture healed time, complications were observed and recovery of shoulder joint function was evaluated by Constant-Murley shoulder score. RESULTS: All the patients were followed up from 12 to 24 weeks with an average of 16 weeks. Operative time was 20 to 40 min with an average of 28 min; intraoperative blood loss was 5 to 25 ml with an average of 10 ml;hospital stay was 3 to 5 days with an average of 3.5 days. All fractures got bone healing and healed time was 12 to 22 weeks with average of 14 weeks. Postoperatively 1 case complicated with wire tail bulging and local irritation symptoms, and the symptoms disappeared when the wire was removed out after the fracture healing; 1 case complicated with local infection after wire tail disengaging, and recovered through anti-septic treatment. According to the Constant-Murley standard of shoulder joint function, 10 cases got excellent results, 15 good, 2 fair, with scores of 89.1±2.7. CONCLUSIONS: Closed reduction and percutaneous double K-wires fixation for the treatment of multisegmental fractures of humeral shaft have advantages of simpler manipulating, less bleeding, less invasive, less complications, and shoulder and elbow joint can obtain good recovery. But closed reduction and percutaneous double K-wires internal fixation cannot be effective against rotation and provide axial stability, the immoblization with a sling or other auxiliary methods should be applied.

[The inactivating effect of chito-oligosaccharides on TMV particles in vitro].

To confirm the inactivating effect of chito-oligosaccharides on Tobacco mosaic virus (TMV) par ticles in vitro, the difference of TMV pathogenicity was evaluated according to the decrease of local lesion numbers after inoculating with TMV mixed with chito-oligosaccharides (DP3-10) in Nicotiana glutinosa, and the virion structural change was studied by transmission electron microscopy after mixed with chito-oligosaccharides. In the range of tested concentrations of chito-oligosaccharides (100-1000 microg /mL), the numbers of local lesions were strongly reduced with over 30% decrement, and the 88.4% reduction gained at the concentration of 600g /mL. It revealed that treatment with chito-oligosaccharide solution of 300-500 microg /mL directly broke TMV particles into tiny pieces of 50-150nm long, and that treatment with solutions of 600-1000 microg/mL caused virus particle agglomerated. The data presented here suggested that chito-oligosaccharides exerted strong inactivating effect on plant virus in vitro.

[Genetic analysis and molecular markers of a novel stripe rust resistance gene YrHua in wheat originated from Psathyrostachys huashanica Keng].

The H9020-17-5,a common wheat-Psathyrostachys huashanica Keng translocation line, possesses excellent resistance to wheat stripe rust. Genetic analysis of F2 and BC1 populations derived from H9020-17-5 x Mingxian169 indicated that resistance to stripe rust in H9020-17-5 was a dominant character controlling by single gene originated from Ps. huashanica. This resistance gene originated from Ps. huashanica was first reported in the present study and named as YrHua. In order to map the resistance gene YrHua, AFLP approach was employed to analyze the 119 individuals of H9020-17-5 x Mingxian169 F2 population which were inoculated by stripe rust isolate CY30. As a result,two markers, PM14(301) and PM42(249) were found to be linked to the resistance gene YrHua,and the genetic distances between the markers and target gene were 5.4 cM and 2.7 cM, respectively. For the convenience of marker-assisted selection in wheat breeding, one of the two AFLP markers was converted to PCR marker using a pair of special primers based on the DNA sequence of PM14 (301) and the polymorphism of restriction site. Our research results provided a useful tool for marker-assisted selection and laid a foundation of fine mapping and map based cloning of YrHua gene.