Stripe rust effector Pst21674 compromises wheat resistance by targeting transcription factor TaASR3.

Pathogens compromise host defense responses by strategically secreting effector proteins. However, the molecular mechanisms by which effectors manipulate disease-resistance factors to evade host surveillance remain poorly understood. In this study, we characterized a Puccinia striiformis f. sp. tritici (Pst) effector Pst21674 with a signal peptide. Pst21674 was significantly upregulated during Pst infections in wheat (Triticum aestivum L.) and knocking down Pst21674 by host-induced gene silencing led to reduced Pst pathogenicity and restricted hyphal spread in wheat. Pst21674 interaction with the abscisic acid-, stress-, and ripening-induced protein TaASR3 was validated mainly in the nucleus. Size exclusion chromatography, bimolecular fluorescence complementation, and luciferase complementation imaging assays confirmed that TaASR3 could form a functional tetramer. Virus-induced gene silencing and overexpression demonstrated that TaASR3 contributes to wheat resistance to stripe rust by promoting accumulation of reactive oxygen species and cell death. Additionally, transcriptome analysis revealed that the expression of defense-related genes was regulated in transgenic wheat plants overexpressing TaASR3. Interaction between Pst21674 and TaASR3 interfered with the polymerization of TaASR3 and suppressed TaASR3-mediated transcriptional activation of defense-related genes. These results indicate that Pst21674 serves as an important virulence factor secreted into the host nucleus to impede wheat resistance to Pst, possibly by targeting and preventing polymerization of TaASR3.

The molecular and functional characterization of ferritins in the hard tick Hyalomma rufipes.

BACKGROUND: The protein ferritin, which plays an important role in the maintenance of iron homeostasis, is indispensable for iron detoxification, resistance to oxidative stress and innate immunity. Ticks, which are obligate blood-sucking ectoparasites, have to deal with a large amount of iron when they take a blood meal. METHODS: Sequence analysis was undertaken using bioinformatics. A recombinant (r) expression vector, rferritin, was constructed for a prokaryotic expression system. A quantitative polymerase chain reaction platform was used to detect the spatial and temporal expression patterns of target genes and their responses to a low temperature environment. Knockdown of the ferritin genes through RNA interference was used to analyze their effects on physiological parameters of ticks. RESULTS: Two ferritin genes, HrFer1 and HrFer2, were cloned from the tick Hyalomma rufipes. Their open reading frames are 519 base pairs (bp) and 573 bp in length, and number of coding amino acids 170 and 190, respectively. The phylogenetic tree showed that HrFer1 and HrFer2 have a close evolutionary relationship with the H subunit of ferritin. In vitro experiments showed that rHrFer1 and rHrFer2 had concentration-dependent iron chelating activity. The relative expression of the two ferritin genes was higher in the ovary and midgut of H. rufipes. RNA interference results demonstrated that HrFer1 and HrFer2 expression had a significant effect on engorged body weight, number of eggs laid, and mortality of H. rufipes, and that HrFer2 also had a significant effect on feeding duration. Furthermore, the relative expression of ferritin decreased significantly in a low temperature environment, suggesting that HrFer1 and HrFer2 play a regulatory role in the cold stress response of H. rufipes. CONCLUSIONS: The results of the present study improve our understanding of the involvement of ferritins in tick blood-feeding.

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.

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.

A Cu-only superoxide dismutase from stripe rust fungi functions as a virulence factor deployed for counter defense against host-derived oxidative stress.

Plants quickly accumulate reactive oxygen species (ROS) to resist against pathogen invasion, while pathogens strive to escape host immune surveillance by degrading ROS. However, the nature of the strategies that fungal pathogens adopt to counteract host-derived oxidative stress is manifold and requires deep investigation. In this study, a superoxide dismutase (SOD) from Puccinia striiformis f. sp. tritici (Pst) PsSOD2 with a signal peptide (SP) and the glycophosphatidyl inositol (GPI) anchor, strongly induced during infection, was analysed for its biological characteristics and potential role in wheat-Pst interactions. The results showed that PsSOD2 encodes a Cu-only SOD and responded to ROS treatment. Heterologous complementation assays in Saccharomyces cerevisiae suggest that the SP of PsSOD2 is functional for its secretion. Transient expression in Nicotiana benthamiana leaves revealed that PsSOD2 is localized to the plasma membrane. In addition, knockdown of PsSOD2 by host-induced gene silencing reduced Pst virulence and resulted in restricted hyphal development and increased ROS accumulation. In contrast, heterologous transient assays of PsSOD2 suppressed flg22-elicited ROS production. Taken together, our data indicate that PsSOD2, as a virulence factor, was induced and localized to the plasma membrane where it may function to scavenge host-derived ROS for promoting fungal infection.

Expression and function assessment of two serpin-type serine protease inhibitors from Haemaphysalis doenitzi.

Serine protease inhibitors (serpins) in ticks are implicated in the modulation of the vertebrate host response to the tick bite. Experimentally, it has been demonstrated that serpins interfere with tick-borne pathogen transmission. However, knowledge on serpins in the tick Haemaphysalis doenitzi is lacking. In this study, the expression of two serpin genes, named HDS1 and HDS2, were assessed in H. doenitzi, and their roles in immune regulation were further investigated. The expression of HDS1 and HDS2 showed no tissue specificity, with maximum expression levels detected in the hemolymph and salivary gland, respectively. Among the developmental stages, the highest expression of HDS1 and HDS2 were detected in larvae and adults, respectively. The recombinant protein rHDS1 displayed obvious inhibitory effects on trypsin and thrombin, whereas rHDS2 clearly inhibited thrombin only. In addition, rHDS1 and rHDS2 showed certain inhibitory activities against bacteria and fungi. The female engorgement body weight, female engorgement rate, and egg hatchability were significantly decreased after injection of double-stranded RNA (dsRNA) of HDS1 gene, whereas no significant effects were observed concerning the feeding period or attachment rate at 24 h after introduction via rabbit ears. When injected with dsRNA of HDS2 gene, no significant effect was observed on the attachment rate at 24 h after introduction into the rabbit ears, but the engorgement body weight and engorgement rate of female ticks were significantly decreased, and no egg hatchment occurred. The above results contribute to better understanding the function of serpins in the development and innate immunity of H. doenitzi.

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.

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.