Sugar transporter TaSTP3 activation by TaWRKY19/61/82 enhances stripe rust susceptibility in wheat.

Sugar efflux from host plants is essential for pathogen survival and proliferation. Sugar transporter-mediated redistribution of host sugar contributes to the outcomes of plant-pathogen interactions. However, few studies have focused on how sugar translocation is strategically manipulated during host colonization. To elucidate this question, the wheat sugar transport protein (STP) TaSTP3 responding to Puccinia striiformis f. sp. tritici (Pst) infection was characterized for sugar transport properties in Saccharomyces cerevisiae and its potential role during Pst infection by RNA interference and overexpression in wheat. In addition, the transcription factors regulating TaSTP3 expression were further determined. The results showed that TaSTP3 is localized to the plasma membrane and functions as a sugar transporter of hexose and sucrose. TaSTP3 confers enhanced wheat susceptibility to Pst, and overexpression of TaSTP3 resulted in increased sucrose accumulation and transcriptional suppression of defense-related genes. Furthermore, TaWRKY19, TaWRKY61 and TaWRKY82 were identified as positive transcriptional regulators of TaSTP3 expression. Our findings reveal that the Pst-induced sugar transporter TaSTP3 is transcriptionally activated by TaWRKY19/61/82 and facilitates wheat susceptibility to stripe rust possibly through elevated sucrose concentration, and suggest TaSTP3 as a strong target for engineering wheat resistance to stripe rust.

First Report of Neofusicoccum parvum Causing Leaf Spot on coral trees (Viburnum odoratissimum) in China.

Coral trees (Viburnum odoratissimum), as a class of evergreen shrubs, are mainly planted in landscapes in numerous cities in China. During September 2020, the author investigated four major parks in Hefei (Bao Park, Hefei Botanical Garden, Luzhou Park and Peninsula Park) and the campus of Anhui Agricultural University (approximately 0.5 ha) (31°49'21.30″N, 117°13'18.25″E). The results showed that the incidence rate of leaf spot disease reached 60% among approximately 100,000 coral trees planted in these areas. Coral trees begin to show leaf spots in August. In early stages of coral trees infection, the symptoms appeared as small brown spots ranged in length from 2 to 3 millimeters on the leaves. After the disease patches expand and darken, the coral leaves eventually wither and fall, which seriously affects its viewing and admiring value. To identify the fungal pathogen, the five-point sampling method was used to take typical similar leaf samples from 5 regions, and 6 samples were taken from each site, so a total of 150 samples were obtained. Fragments of sample leaves were surface-sterilized with 1% NaClO, plated on potato dextrose agar, and incubated at 25 °C in the dark. A total of 275 strains were obtained from 150 samples. According to the morphological characteristics, 275 strains were purified and divided into four types. Four representative strains (MI1, K1, F1, D1) were selected from four types for further pathogenicity testing and identification. The pathogenicity test was conducted in triplicate by inoculating wounded leaves of 1-year-old potted V. odoratissimum with 20µL of a conidial suspension (106 conidia/mL). The control was inoculated with sterile water. The specimens were placed in a growth chamber while maintaining 90% relative humidity and 28℃. After five days, the characteristic lesions were observed only on inoculated MI1 spore suspension leaves. The same fungus was reisolated from the lesions, thus fulfilling Koch's postulates. The pathogenic fungi accounted for 60% of all strains. Fungal colonies were circular and had abundant white aerial mycelium, and colonies changed from white to pure black after maturity. Conidia were fusiform (16-17×5-6 µm), thin-walled, transparent, and without diaphragms. Molecular identification was performed by partially sequencing the internal transcribed spacer (ITS) gene, the translation elongation Factor 1-alpha(EF1-alpha) gene, and the ß-tubulin (TUB2) gene by using the primers ITS1/ITS4 (White et al. 1990), EF1-728F (Alves et al. 2008)/EF1-986R (Carbone & Kohn 1999), and Bt2a/Bt2b (Glass & Donaldson 1995), respectively. The obtained ITS sequence (MW767713) showed 99% identity with N. parvum CMW28429 (KU997429.1), the EF1-alpha sequence (MZ398261) showed 99% identity with N. parvum isolate A4 (FJ528597.1), and the TUB2 sequence (MZ398260) showed 99% identity with N. parvum isolate BO52 (KU554657.1). By combining the sequences of individual fragments of each fungus in the order ITS, EF1-alpha and TUB2, MEGA 6.0 was used to analyze the sequence of kinship by using the maximum likelihood method, and the repeat value of bootstraps was 1000. A polygenic phylogenetic tree analysis based on multilocus alignment (ITS, EF1-alpha and TUB2) was constructed with some strains of Botryosphaeriaceae species. The results of the phylogenetic tree showed that MI1 and N. parvum clustered into a branch. To our knowledge, this is the first report of N. parvum causing leaf spot on V. odoratissimum in China.

AtSTP8, an endoplasmic reticulum-localised monosaccharide transporter from Arabidopsis, is recruited to the extrahaustorial membrane during powdery mildew infection.

Biotrophic pathogens are believed to strategically manipulate sugar transport in host cells to enhance their access to carbohydrates. However, mechanisms of sugar translocation from host cells to biotrophic fungi such as powdery mildew across the plant-haustorium interface remain poorly understood. To investigate this question, systematic subcellular localisation analysis was performed for all the 14 members of the monosaccharide sugar transporter protein (STP) family in Arabidopsis thaliana. The best candidate AtSTP8 was further characterised for its transport properties in Saccharomyces cerevisiae and potential role in powdery mildew infection by gene ablation and overexpression in Arabidopsis. Our results showed that AtSTP8 was mainly localised to the endoplasmic reticulum (ER) and appeared to be recruited to the host-derived extrahaustorial membrane (EHM) induced by powdery mildew. Functional complementation assays in S. cerevisiae suggested that AtSTP8 can transport a broad spectrum of hexose substrates. Moreover, transgenic Arabidopsis plants overexpressing AtSTP8 showed increased hexose concentration in leaf tissues and enhanced susceptibility to powdery mildew. Our data suggested that the ER-localised sugar transporter AtSTP8 may be recruited to the EHM where it may be involved in sugar acquisition by haustoria of powdery mildew from host cells in Arabidopsis.

TaSTP13 contributes to wheat susceptibility to stripe rust possibly by increasing cytoplasmic hexose concentration.

BACKGROUND: Biotrophic fungi make intimate contact with host cells to access nutrients. Sugar is considered as the main carbon sources absorbed from host cells by pathogens. Partition, exchanges and competition for sugar at plant-pathogen interfaces are controlled by sugar transporters. Previous studies have indicated that the leaf rust resistance (Lr) gene Lr67, a natural mutation of TaSTP13 encoding a wheat sugar transport protein, confers partial resistance to all three wheat rust species and powdery mildew possibly due to weakened sugar transport activity of TaSTP13 by heterodimerization. However, one major problem that remains unresolved concerns whether TaSTP13 participates in wheat susceptibility to rust and mildew. RESULTS: In this study, expression of TaSTP13 was highly induced in wheat leaves challenged by Puccinia striiformis f. sp. tritici (Pst) and certain abiotic treatments. TaSTP13 was localized in the plasma membrane and functioned as homooligomers. In addition, a functional domain for its transport activity was identified in yeast. Suppression of TaSTP13 reduced wheat susceptibility to Pst by barley stripe mosaic virus-induced gene silencing (VIGS). While overexpression of TaSTP13 promoted Arabidopsis susceptibility to powdery mildew and led to increased glucose accumulation in the leaves. CONCLUSIONS: These results indicate that TaSTP13 is transcriptionally induced and contributes to wheat susceptibility to stripe rust, possibly by promoting cytoplasmic hexose accumulation for fungal sugar acquisition in wheat-Pst interactions.

A stripe rust effector Pst18363 targets and stabilises TaNUDX23 that promotes stripe rust disease.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), poses a tremendous threat to the production of wheat worldwide. The molecular mechanisms of Pst effectors that regulate wheat immunity are poorly understood. In this study, we identified an effector Pst18363 from Pst that suppresses plant cell death in Nicotiana benthamiana and in wheat. Knocking down Pst18363 expression by virus-mediated host-induced gene silencing significantly decreased the number of rust pustules, indicating that Pst18363 functions as an important pathogenicity factor in Pst. Pst18363 was proven to interact with wheat Nudix hydrolase 23 TaNUDX23. In wheat, silencing of TaNUDX23 by virus-induced gene silencing increased reactive oxygen species (ROS) accumulation induced by the avirulent Pst race CYR23, whereas overexpression of TaNUDX23 suppressed ROS accumulation induced by flg22 in Arabidopsis. In addition, TaNUDX23 suppressed Pst candidate effector Pst322-trigged cell death by decreasing ROS accumulation in N. benthamiana. Knocking down of TaNUDX23 expression attenuated Pst infection, indicating that TaNUDX23 is a negative regulator of defence. In N. benthamiana, Pst18363 stabilises TaNUDX23. Overall, our data suggest that Pst18363 stabilises TaNUDX23, which suppresses ROS accumulation to facilitate Pst infection.

ABA-Induced Sugar Transporter TaSTP6 Promotes Wheat Susceptibility to Stripe Rust.

Biotrophic pathogens, such as wheat rust fungi, survive on nutrients derived from host cells. Sugar appears to be the major carbon source transferred from host cells to various fungal pathogens; however, the molecular mechanism by which host sugar transporters are manipulated by fungal pathogens for nutrient uptake is poorly understood. TaSTP6, a sugar transporter protein in wheat (Triticum aestivum), was previously shown to exhibit enhanced expression in leaves upon infection by Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. In this study, we found that Pst infection caused increased accumulation of abscisic acid (ABA) and that application of exogenous ABA significantly enhanced TaSTP6 expression. Moreover, knockdown of TaSTP6 expression by barley stripe mosaic virus-induced gene silencing reduced wheat susceptibility to the Pst pathotype CYR31, suggesting that TaSTP6 expression upregulation contributes to Pst host sugar acquisition. Consistent with this, TaSTP6 overexpression in Arabidopsis (Arabidopsis thaliana) promoted plant susceptibility to powdery mildew and led to increased Glc accumulation in the leaves. Functional complementation assays in Saccharomyces cerevisiae showed that TaSTP6 has broad substrate specificity, indicating that TaSTP6 is an active sugar transporter. Subcellular localization analysis indicated that TaSTP6 localizes to the plasma membrane. Yeast two-hybrid and bimolecular fluorescence complementation experiments revealed that TaSTP6 undergoes oligomerization. Taken together, our results suggest that Pst stimulates ABA biosynthesis in host cells and thereby upregulates TaSTP6 expression, which increases sugar supply and promotes fungal infection.

A novel citrate synthase isoform contributes infection and stress resistance of the stripe rust fungus.

The early development of a rust fungus is dependent on the endogenous lipids stored in the urediniospores. After it establishes a parasitic relationship with the host, sugars absorbed from the host cells by haustoria become the primary nutrients. The tricarboxylic acid (TCA) cycle is essential to oxidize these nutrients. However, few studies have addressed the role of citrate synthase (CS), a rate-limiting enzyme of the TCA cycle, during the infection process of rust fungi. In this study, a CS gene from Puccinia striiformis f. sp. tritici (Pst), PsCS1, was cloned and characterized. Transcripts of PsCS1 and the enzyme activity of the CS were increased in the early Pst infection stage. Biochemical features and subcellular localization revealed that PsCS1 encoded a mitochondrial CS. Size exclusion chromatography, yeast two-hybrid and bimolecular fluorescence complementation experiments confirmed that PsCS1 could form a functional homo-octamer. The overexpression of PsCS1 enhanced the resistance of Escherichia coli to salt stress. The knockdown of PsCS1 using a host-induced gene silencing (HIGS) system blocked Pst growth in wheat. These results indicate that PsCS1 is required for nutrient metabolism in Pst and contributes to Pst infection by regulating ATP production and the supply of carbon sources.

A unique invertase is important for sugar absorption of an obligate biotrophic pathogen during infection.

An increased invertase activity in infected plant tissue has been observed in many plant-pathogen interactions. However, the origin of this increased invertase activity (plant and/or pathogen) is still under debate. In addition, the role of pathogen invertases in the infection process is also unclear. We identified and cloned a gene with homology to invertases from Puccinia striiformis f. sp. tritici (Pst). Transcript levels of PsINV were analyzed by quantitative reverse transcription PCR in both compatible and incompatible Pst-wheat interactions . Function of the gene product was confirmed by heterologous expression, and its function in Pst infection was analyzed by host-induced gene silencing (HIGS). Pst abundantly secretes invertase during its invasion attempts whether in a compatible or incompatible interaction with wheat. Further research into the different domains of this protein indicated that the rust-specific sequence contributes to a higher efficiency of sucrose hydrolysis. With PsINV silenced by HIGS during the infection process, growth of Pst is inhibited and conidial fructification incomplete. Finally, pathogenicity of Pst is impaired and spore yield significantly reduced. Our results clearly demonstrate that this Pst invertase plays a pivotal role in this plant-pathogen interaction probably by boosting sucrose hydrolysis to secure the pathogen's sugar absorption.

An extracellular Zn-only superoxide dismutase from Puccinia striiformis confers enhanced resistance to host-derived oxidative stress.

Accumulation of reactive oxygen species (ROS) following plant-pathogen interactions can trigger plant defence responses and directly damage pathogens. Thus, it is essential for pathogens to scavenge host-derived ROS to establish a parasitic relationship. However, the mechanisms protecting pathogens from host-derived oxidative stress remain unclear. In this study, a superoxide dismutase (SOD) gene, PsSOD1, was cloned from a wheat-Puccinia striiformis f. sp. tritici (Pst) interaction cDNA library. Transcripts of PsSOD1 were up-regulated in the early infection stage. Heterologous mutant complementation and biochemical characterization revealed that PsSOD1 encoded a Zn-only SOD. The predicted signal peptide was functional in an invertase-mutated yeast strain. Furthermore, immunoblot analysis of apoplastic proteins in Pst-infected wheat leaves and bimolecular fluorescence complementation suggested that PsSOD1 is a secreted protein that potentially forms a dimer during Pst infection. Overexpression of PsSOD1 enhanced Schizosaccharomyces pombe resistance to exogenous superoxide. Transient expression of PsSOD1 in Nicotiana benthamiana suppressed Bax-induced cell death. Knockdown of PsSOD1 using a host-induced gene silencing (HIGS) system reduced the virulence of Pst, which was associated with ROS accumulation in HIGS plants. These results suggest that PsSOD1 is an important pathogenicity factor that is secreted into the host-pathogen interface to contribute to Pst infection by scavenging host-derived ROS.

Down-regulation of a wheat alkaline/neutral invertase correlates with reduced host susceptibility to wheat stripe rust caused by Puccinia striiformis.

Numerous studies have found that sucrose (Suc) metabolism plays a crucial role in the environmental stress response of many plant species. The majority of Suc metabolism-associated reports refer to acid invertases (Ac-Invs). However, alkaline/neutral Invs (A/N-Invs) have been poorly studied. In this study, a wheat A/N-Inv gene, Ta-A/N-Inv1, with three copies located on chromosomes 4A, 4B, and 4D, was cloned from a wheat-Puccinia striiformis f. sp. tritici (Pst) interaction cDNA library. Transcripts of the three Ta-A/N-Inv1 copies were up-regulated in wheat leaves that were infected by Pst or had experienced certain abiotic treatments. Furthermore, the expression of Ta-A/N-Inv1 was decreased by treatment with exogenous hormones. Heterologous mutant complementation and subcellular localization revealed that Ta-A/N-Inv1 is a cytoplasmic invertase. Knocking down all three copies of Ta-A/N-Inv1 using the barley stripe mosaic virus-induced gene silencing system reduced the susceptibility of wheat to the Pst virulent pathotype CYR31, which is associated with pathogen-induced H2O2 accumulation and enhanced necrosis. Interestingly, 48h dark treatment of the Ta-A/N-Inv1-knockdown plants immediately after inoculation abrogated their enhanced resistance, suggesting that H2O2 production and its associated cell death and resistance in the Ta-A/N-Inv1-silenced plants require light. Consistent with this observation, photosynthesis and reactive oxygen species (ROS)-related genes were significantly up-regulated in the Ta-A/N-Inv1-knockdown plants infected by CYR31 under light exposure. These results suggest that Ta-A/N-Inv1 might act as a negative regulator in wheat disease resistance to Pst by increasing cytoplasmic hexose accumulation and downregulating photosynthesis of the leaves to avoid cell death due to excessive ROS production.

Priming transcranial direct current stimulation for improving hemiparetic upper limb in patients with subacute stroke: study protocol for a randomised controlled trial.

INTRODUCTION: Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that modulates brain states by applying a weak electrical current to the brain cortex. Several studies have shown that anodal stimulation of the ipsilesional primary motor cortex (M1) may promote motor recovery of the affected upper limb in patients with stroke; however, a high-level clinical recommendation cannot be drawn in view of inconsistent findings. A priming brain stimulation protocol has been proposed to induce stable modulatory effects, in which an inhibitory stimulation is applied prior to excitatory stimulation to a brain area. Our recent work showed that priming theta burst magnetic stimulation demonstrated superior effects in improving upper limb motor function and neurophysiological outcomes. However, it remains unknown whether pairing a session of cathodal tDCS with a session of anodal tDCS will also capitalise on its therapeutic effects. METHODS AND ANALYSIS: This will be a two-arm double-blind randomised controlled trial involving 134 patients 1-6 months after stroke onset. Eligible participants will be randomly allocated to receive 10 sessions of priming tDCS+robotic training, or 10 sessions of non-priming tDCS+robotic training for 2 weeks. The primary outcome is the Fugl-Meyer Assessment-upper extremity, and the secondary outcomes are the Wolf Motor Function Test and Modified Barthel Index. The motor-evoked potentials, regional oxyhaemoglobin level and resting-state functional connectivity between the bilateral M1 will be acquired and analysed to investigate the effects of priming tDCS on neuroplasticity. ETHICS AND DISSEMINATION: The study has been approved by the Research Ethics Committee of the Shanghai Yangzhi Rehabilitation Center (reference number: Yangzhi2023-022) and will be conducted in accordance with the Declaration of Helsinki of 1964, as revised in 2013. TRIAL REGISTRATION NUMBER: ChiCTR2300074681.

Cortical mapping of active and passive upper limb training in stroke patients and healthy people: A functional near-infrared spectroscopy study.

Active exercise for upper limb training has been widely used to improve hemiplegic upper limb function, and its effect may be boosted by extrinsic visual feedback. The passive movement of the hemiplegic upper limb is also commonly used. We conducted a functional near-infrared spectroscopy experiment to compare cortical activation during the following three conditions: active left upper limb movement (on the hemiplegic sides in stroke patients), with or without extrinsic motor performance visual feedback (LAV, LAnV), and passive left upper limb movement (hemiplegic sides in stroke patients) (LP) in stroke patients and healthy controls. Twenty patients with right hemispheric stroke and 20 healthy controls were recruited for this study. Hemodynamic changes were detected during left upper limb movements (on the hemiplegic sides in stroke patients) under the above three conditions in the sensorimotor cortex (SMC), supplementary motor area (SMA), and premotor cortex (PMC). There was no significant difference in the level of cortical activation between patients with stroke and healthy subjects during the three conditions. Both the LAV and LAnV induced significantly higher activation in the contralateral SMA and PMC than in the LP. Extrinsic visual feedback led to additional activation in the contralateral PMC and SMA, but this was not statistically significant. Our study indicates that active upper-limb movement appears to induce higher cortical activation than that elicited by passive movement in both stroke patients and the healthy population. Extrinsic motor performance in the form of visual feedback provided during active movement may facilitate sensorimotor areas over the contralateral hemisphere.

Icaritin Preparation from Icariin by a Special Epimedium Flavonoid-Glycosidase from Aspergillus sp.y848 Strain.

In this study, to obtain icaritin with high pharmacological activities from icariin, which has a content ratio of over 58% in the total flavonoids of Epimedium herb, a special Epimedium flavonoid-glycosidase was produced, purified and characterized from Aspergillus sp.y848 strain. The optimal enzyme production was gained in a medium containing 5% (w/v) wheat bran extract and 0.7% (w/v) Epimedium leaf powder as the enzyme inducer, and strain culture at 30°C for 6-7 days. The molecular weight of the enzyme was approximately 73.2 kDa; the optimal pH and temperature were 5.0 and 40°C. The enzyme Km and Vmax values for icariin were 15.63 mM and 55.56 mM/h. Moreover, the enzyme hydrolyzed the 7-O-glucosides of icariin into icariside II, and finally hydrolyzed 3-O-rhamnoside of icariside II into icaritin. The enzyme also hydrolyzed 7-O-glucosides of epimedin B to sagittatoside B, and then further hydrolyzed terminal 3-O-xyloside of sagittatoside B to icarisiede II, before finally hydrolyzing 3-O-rhamnoside of icarisiede II into icaritin. The enzyme only hydrolyzed 7-O-glucoside of epimedin A or epimedin C into sagittatoside A or sagittatoside C. It is possible to prepare icaritin from the high-content icariin in Epimedium herb using this enzyme. When 2.5% icariin was reacted at 40°C for 18-20 h by the low-cost crude enzyme, 5.04 g icaritin with 98% purity was obtained from 10 g icariin. Also, the icaritin molar yield was 92.5%. Our results showed icaritin was successfully produced via cost-effective and relatively simple methods from icariin by crude enzyme. Our results should be very useful for the development of medicines from Epimedium herb.

TaRar1 Is Involved in Wheat Defense against Stripe Rust Pathogen Mediated by YrSu.

RAR1 is a eukaryotic zinc-binding protein first identified as required for race-specific resistance to powdery mildew in barley. To study the function of TaRAR1 involvement in wheat (Triticum aestivum L.) defense against the infection of stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst), we identified and cloned three wheat homeologous genes highly similar to the barley HvRar1, designated as TaRar1-2A, TaRar1-2B, and TaRar1-2D. The three TaRAR1 proteins all contain two conserved cysteine-and histidine-rich domains (CHORD-I and -II) shared by known RAR1-like proteins. Characterization of TaRar1 expression revealed that the expression was tissue-specific and up-regulated in wheat during stripe rust infection. Moreover, the transcription of TaRar1 was induced by methyl jasmonate, ethylene, and abscisic acid hormones. The same results were observed with drought and wound treatments. After TaRar1 was silenced in wheat cultivar Suwon11 containing the stripe rust resistance gene YrSu, the endogenous salicylic acid (SA) level, the hydrogen peroxide (H2O2) accumulation and the degree of hypersensitive response (HR) were significantly decreased, and the resistance to the avirulent pathotype of stripe rust was compromised. Meanwhile, the expression of catalase, an enzyme required for H2O2-scavenging, was up-regulated. Taken together, we concluded that TaRar1 is involved in wheat defense against stripe rust mediated by YrSu, and the defense was through SA to influence reactive oxygen species accumulation and HR.