Specific microRNA/mRNA expression profiles and novel immune regulation mechanisms are induced in THP-1 macrophages by in vitro exposure to Trichosporon asahii.

BACKGROUND: Trichosporon asahii is considered the most prominent species associated with invasive trichosporonosis, but little is known about the pathogenesis of T. asahii infection in the host. MicroRNAs (miRNAs) are a class of noncoding endogenous small RNAs that play vital roles by manipulating immune responses against pathogenic microorganisms. Nevertheless, the exact functions of miRNAs in T. asahii infection are still unknown. OBJECTIVE: To investigate the interactions involved in the miRNA immune response in THP-1 macrophages following in vitro exposure to T. asahii. METHODS: We utilized next-generation sequencing to detect differentially expressed (DE) miRNAs and mRNAs in THP-1 cells after 24 h of in vitro exposure to T. asahii. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to verify the sequencing results. The miRNA-mRNA regulatory network was constructed with the DE miRNAs and DE mRNAs. We performed Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of the predicted targeting mRNAs in the miRNA-mRNA network. A dual-luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA) were utilized to demonstrate the reliability of the miR-342-3p/Dectin-1 pair. RESULTS: A total of 120 DE miRNAs and 588 DE mRNAs were identified after 24 h of in vitro exposure to T. asahii. The miRNA-mRNA regulatory network was constructed with 39 DE miRNAs and 228 DE mRNAs. KEGG pathway analysis revealed that the up-regulated DE mRNAs in the complex interaction network were mainly involved in immune-related pathways. In addition, we verified the target relationship between miR-342-3p and Dectin-1 and found that miR-342-3p could promote the expression of TNF-α and IL-6 by negatively regulating Dectin-1. CONCLUSIONS: This study evaluated the expression profiles of miRNA/mRNA and revealed the immunological consequences of THP-1 macrophages in response to T. asahii exposure. Moreover, our data suggest that miR-342-3p can indirectly promote inflammatory responses and may be a potential therapeutic target against trichosporonosis.

Prominence of IL6, IGF, TLR, and Bioenergetics Pathway Perturbation in Lung Tissues of Scleroderma Patients With Pulmonary Fibrosis.

Scleroderma-associated pulmonary fibrosis (SSc-PF) and idiopathic pulmonary fibrosis (IPF) are two of many chronic fibroproliferative diseases that are responsible for nearly 45% of all deaths in developed countries. While sharing several pathobiological characteristics, they also have very distinct features. Currently no effective anti-fibrotic treatments exist that can halt the progression of PF or reverse it. Our goal is to uncover potential gene targets for the development of anti-fibrotic therapies efficacious in both diseases, and those specific to SSc-PF, by identifying universal pathways and molecules driving fibrosis in SSc-PF and IPF tissues as well as those unique to SSc-PF. Using DNA microarray data, a meta-analysis of the differentially expressed (DE) genes in SSc-PF and IPF lung tissues (diseased vs. normal) was performed followed by a full systems level analysis of the common and unique transcriptomic signatures obtained. Protein-protein interaction networks were generated to identify hub proteins and explore the data using the centrality principle. Our results suggest that therapeutic strategies targeting IL6 trans-signaling, IGFBP2, IGFL2, and the coagulation cascade may be efficacious in both SSc-PF and IPF. Further, our data suggest that the expression of matrikine-producing collagens is also perturbed in PF. Lastly, an overall perturbation of bioenergetics, specifically between glycolysis and fatty acid metabolism, was uncovered in SSc-PF. Our findings provide insights into potential targets for the development of anti-fibrotic therapies that could be effective in both IPF and SSc-PF.

An effector protein of the wheat stripe rust fungus targets chloroplasts and suppresses chloroplast function.

Chloroplasts are important for photosynthesis and for plant immunity against microbial pathogens. Here we identify a haustorium-specific protein (Pst_12806) from the wheat stripe rust fungus, Puccinia striiformis f. sp. tritici (Pst), that is translocated into chloroplasts and affects chloroplast function. Transient expression of Pst_12806 inhibits BAX-induced cell death in tobacco plants and reduces Pseudomonas-induced hypersensitive response in wheat. It suppresses plant basal immunity by reducing callose deposition and the expression of defense-related genes. Pst_12806 is upregulated during infection, and its knockdown (by host-induced gene silencing) reduces Pst growth and development, likely due to increased ROS accumulation. Pst_12806 interacts with the C-terminal Rieske domain of the wheat TaISP protein (a putative component of the cytochrome b6-f complex). Expression of Pst_12806 in plants reduces electron transport rate, photosynthesis, and production of chloroplast-derived ROS. Silencing TaISP by virus-induced gene silencing in a susceptible wheat cultivar reduces fungal growth and uredinium development, suggesting an increase in resistance against Pst infection.

Liver abscess due to Sterigmatomyces halophilus in a boy with acute lymphoblastic leukemia.

We report the first case of liver abscess due to Sterigmatomyces halophilus. Because this pathogen grows poorly in culture medium without added salts, it was identified by sequencing analysis targeting the rRNA gene internal transcribed spacer (ITS) region. This method could be useful for pathogens that cannot be cultured using standard methods.

A unique polysaccharide from Hericium erinaceus mycelium ameliorates acetic acid-induced ulcerative colitis rats by modulating the composition of the gut microbiota, short chain fatty acids levels and GPR41/43 respectors.

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colonic mucosa. Risk of colorectal cancer in ulcerative colitis is increased in patients with long-standing disease compared with the general population. Hericium erinaceus (HE) has been used in traditional folk medicine and medicinal cuisine in China, Korea and Japan with anti-gastritis and anti-ulcerative colitis activities. EP-1, a purified unique polysaccharide isolated from HE mycelium, has recently been identified as the active component responsible for anti- ulcerative colitis activity by using a cell model for identification. In this study, our data shows that EP-1 was effective in relieving the symptoms of acetic acid induced UC rats. Based on the Illumina MiSeq platform, 16S rRNA sequencing of the rat colonic contents indicated that the intestinal flora structure remarkably changed in the model rats and the tendency was alleviated to a certain degree by EP-1. The further results showed that in the acetic acid induced UC rats EP-1 modulated the gut microbiota community and increased short chain fatty acids (SCFAs). And immunoblot analyses showed that after treated by EP-1, GPR41 and GPR43 were significantly suppressed expression in colonic tissues of the UC rats. In the meanwhile, EP-1 also showed its antioxidant, anti-inflammatory and enhancing immune activities. Thus, the polysaccharide purified from HE showed potential for anti-UC activity and the complementary and alternative medicine (CAM) herb therapy.

Hericium erinaceus isolectins recognize mucin-type O-glycans as tumor-associated carbohydrate antigens on the surface of K562 human leukemia cells.

In Hericium erinaceus mushroom fruiting body, two different lectin groups, HEL1 and HEL2, were identified by using peptide mass fingerprinting based on customized protein sequence databases derived from RNA-Seq data. The HEL2 group included four isoforms designated HEL2a-d. Codon-optimized genes encoding HEL1, HEL2a, and HEL2b were expressed in Escherichia coli to produce fully active soluble proteins designated rHEL1, rHEL2a, and rHEL2b. Interestingly, these lectins showed different molecular weights: approximately 15 kDa for rHEL1 and approximately 120 kDa for rHEL2a and rHEL2b under non-denaturing conditions. rHEL2a and rHEL2b exhibited agglutination activities, but rHEL1 did not show any agglutination activity toward animal erythrocytes. The hemagglutination activity of rHEL2 lectins was strongly inhibited by glycoproteins containing mucin-type O-glycans. Glycan array analysis and isothermal titration calorimetry revealed that rHEL2 isolectins interacted strongly with O-glycans harboring the core 1 O-glycan motif, Galß(1,3)GalNAc. Moreover, the glycan binding specificities of rHEL2 isolectins were comparable to that of peanut agglutinin in their ability to recognize O-glycans attached to leukosialin as tumor-associated carbohydrate antigens on the surface of K562 human leukemia cells. These results indicate that rHEL2 isolectins could be used as a powerful tool for analyzing mucin-type O-glycans expressed on the surface of cancer cells.

A Puccinia striiformis f. sp. tritici secreted protein activates plant immunity at the cell surface.

Pathogens secrete effector proteins to suppress host immunity, mediate nutrient uptake and subsequently enable parasitism. However, on non-adapted hosts, effectors can be detected as non-self by host immune receptors and activate non-host immunity. Nevertheless, the molecular mechanisms of effector triggered non-host resistance remain unknown. Here, we report that a small cysteine-rich protein PstSCR1 from the wheat rust pathogen Puccinia striiformis f. sp. tritici (Pst) activates immunity in the non-host solanaceous model plant Nicotiana benthamiana. PstSCR1 homologs were found to be conserved in Pst, and in its closest relatives, Puccinia graminis f. sp. tritici and Puccinia triticina. When PstSCR1 was expressed in N. benthamiana with its signal peptide, it provoked the plant immune system, whereas no stimulation was observed when it was expressed without its signal peptide. PstSCR1 expression in N. benthamiana significantly reduced infection capacity of the oomycete pathogens. Moreover, apoplast-targeted PstSCR1 triggered plant cell death in a dose dependent manner. However, in Brassinosteroid insensitive 1-Associated Kinase 1 (SERK3/BAK1) silenced N. benthamiana, cell death was remarkably decreased. Finally, purified PstSCR1 protein activated defence related gene expression in N. benthamiana. Our results show that a Pst-secreted protein, PstSCR1 can activate surface mediated immunity in non-adapted hosts and contribute to non-host resistance.

Activation of NADPH Oxidase by ß-Glucan from Phellinus baumii (Agaricomycetes) in RAW 264.7 Cells.

Production of oxygen-derived free radicals in phagocytes is important in preventing bacterial and fungal infections. Among free radicals, superoxide anions are a typical reactive oxygen species secreted by macrophages and neutrophils. NADPH oxidase (NOX) is a key producer of superoxide anions in these cells. ß-glucans from mushrooms modulate the immune system by binding with the dectin-1 receptor on macrophages. Dectin-1 functions as a pattern recognition receptor that recognizes the pathogen-associated molecular pattern of ß-glucans. During dectin-1 signaling, NOX functions in the activated macrophages to produce ROS, which are critical in antimicrobial host defense. In this study, NOX activation was measured using a lucigenin chemiluminescence assay in RAW 264.7 murine macrophages treated for 1 hour with a ß-glucan fraction from Phellinus baumii (BGF; 10, 100, 500, and 1000 µg/mL) in the absence or presence of phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). NOX was activated at BGF concentrations exceeding 10 µg/mL. BGF in the presence of PMA or LPS activated the enzyme more than treatment with PMA or LPS alone. In the presence of the NOX inhibitor diphenyleneiodonium, BGF still activated NOX. When macrophages were treated with BGF and Staphylococcus aureus, bacterial viability was reduced in a concentration-dependent manner, possibly as a result of increased phagocytosis and oxygen radical production by the activated NOX. These results demonstrate that BGF is a potent stimulator of NOX in macrophages and augments macrophage-mediated phagocytosis and NOX activity.

Protein-bound polysaccharides from Coriolus versicolor attenuate LPS-induced synthesis of pro-inflammatory cytokines and stimulate PBMCs proliferation.

Protein-bound polysaccharides (PBP) isolated from Coriolus versicolor (CV) are classified as biological response modifiers capable of exhibiting various biological activities, such as anti-tumour and immunopotentiating activity. Since we have found in vivo studies that the tested PBP induced prolongation of endotoxin fever in rats, the aim of the present study was to investigate the in vitro effect of the PBP on the production of pro-inflammatory cytokines by the lipolysaccharide (LPS)-stimulated rat peripheral blood mononuclear cells (PBMCs). The results showed that the PBP affect the immunomodulating properties of the LPS-treated PBMCs by the enhancement of mitogenic activity and attenuation of the LPS-induced production of interleukin (IL)-1ß and IL-6. Moreover, the tested polysaccharides peptides themselves also exhibit immunomodulatory properties manifested in the increased cell proliferation and pro-inflammatory cytokine release from PBMCs. The effect of PBP on the both phenomena was time-dependent and occurred in the U-shaped dose response manner. These findings are significant when considering the use of commercially available PBP from CV extract by cancer patients suffering from immunodeficiency, who may experience microbial infections during therapy.

Molecular cloning and function characterization of a new macrophage-activating protein from Tremella fuciformis.

Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1ß, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway.

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity.

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

A physiologically regulated multidomain cystatin of wheat shows stage-dependent immunity against Karnal Bunt (Tilletia indica).

To identify novel components of basal resistance against the Tellitia indica of wheat, breeding for disease resistance was carried out on resistant and susceptible genotype of Karnal Bunt. The different members of wheat cystatin gene families were cloned, and their role in triggering differential resistance through co-expression was analyzed in our lab. The multidomain wheat cystatin (WCM) is a proteinase inhibitor characterized by cloning the gene from susceptible (WH542) and resistant genotype (HD 29). A WCM cDNA was isolated from both genotypes and sequenced. The WCM had a highly conserved N-terminal cystatin domain and a long C-terminal extension containing a second region, which exhibited similarity to the cystatin domain. The expression level was significantly (P > 0.001) higher in resistant compared to susceptible genotype at all the physiological stages of wheat spikes. In order to characterize the biochemical properties of WCM, the coding sequence was expressed in Escherichia coli using pET expression vector. The recombinant WCM was purified from soluble fraction of the cell extract by using affinity chromatography. WCM, with 23 KDa molecular mass, showed cysteine proteinase inhibitory activity against papain (Ki 3.039 × 10(-7) M) as determined by using BAPNA as substrate. Furthermore, it was able to arrest the fungal mycelial growth of T. indica. Hyphae growth was inhibited, and morphological changes such as swelling and fragmentation of the fungus were observed. Overall, these observations suggest an endogenous high expression of cystatin, possibly associated with the resistance of wheat against Karnal bunt.

Haplotype structure around Bru1 reveals a narrow genetic basis for brown rust resistance in modern sugarcane cultivars.

Modern sugarcane cultivars (Saccharum spp., 2n = 100-130) are high polyploid, aneuploid and of interspecific origin. A major gene (Bru1) conferring resistance to brown rust, caused by the fungus Puccinia melanocephala, has been identified in cultivar R570. We analyzed 380 modern cultivars and breeding materials covering the worldwide diversity with 22 molecular markers genetically linked to Bru1 in R570 within a 8.2 cM segment. Our results revealed a strong LD in the Bru1 region and strong associations between most of the markers and rust resistance. Two PCR markers, that flank the Bru1-bearing segment, were found completely associated with one another and only in resistant clones representing efficient molecular diagnostic for Bru1. On this basis, Bru1 was inferred in 86 % of the 194 resistant sugarcane accessions, revealing that it constitutes the main source of brown rust resistance in modern cultivars. Bru1 PCR diagnostic markers should be particularly useful to identify cultivars with potentially alternative sources of resistance to diversify the basis of brown rust resistance in breeding programs.

Immunostimulatory properties of a polysaccharide isolated from Astraeus hygrometricus.

Mushrooms have been used as food and for therapeutic purpose for decades, and various compounds derived from these have potential biological activities. Application of such agents of natural origin is vital with relevance to an increase in diseases involving immune dysfunction, cancer, and autoimmune conditions in recent years. These compounds belong mainly to polysaccharides, especially beta-D-glucan, glycopeptide/protein complexes, proteoglycans, proteins, and triterpenoids. Among these compounds, polysaccharides and their peptide/protein derivates have extremely important roles in immunomodulating activities. The present study aims to explore the immunomodulatory properties of polysaccharide isolated from the mushroom Astraeus hygrometricus. The polysaccharide isolated by hot alkaline extraction and chromatography was designated as AE2 and studied for its immunostimulatory potential in vivo in a murine model. Macrophages from treated mice showed higher production of nitric oxide and interleukin-1 and also exhibited an increased phagocytic potential. It also enhanced natural killer cell activation and proliferation of splenocytes with an increase in the T-helper (Th) 1 cytokine level of splenocyte culture supernatant. The cytokine level in serum also indicated a Th1 skew. The observed activities were associated with no general toxicity to the organism. The findings will be helpful in considering A. hygrometricus as a potential source of an immunomodulator and in designing further studies to understand its mode of action on immune system.

Antineoplasic activity of Agaricus brasiliensis basidiocarps on different maturation phases

The fungus Agaricus brasiliensis is a Basidiomycete studied because of its immunomodulation and/or antitumor substances. The objective of this study was to verify the Agaricus brasiliensis antineoplasic activity in vivo on different basidiocarp maturation phases on Sarcoma 180 cells implanted in mice. Sarcoma cells were implanted in mice and after seven days mice were divided in three groups. The first group was treated with saline solution, the second group was treated with closed basidiocarp extract solution and the third group was treated with opened basidiocarp extract solution. After 30 days of being daily orally treated with these three solutions all animals suffered euthanasia, and the splenic index, tumor mass and volume were determined. No significant differences of the tumor growth inhibition in function of the different basidiocarp maturation phases for the Agaricus brasiliensis strain were observed. The in vivo basidiocarp antineoplasic average activity was 89.22 percent.

Surface plasmon resonance immunosensor for early diagnosis of Asian rust on soybean leaves.

Soybean rust (Asian rust) is a disease that occurs in soy cultures, negatively affecting pod formation and final grain weight and reducing value and product quality. Early identification of fungus in the plants prevents severe farming losses and spread to neighboring cultures. In this paper, a fast response sensor was developed based on surface plasmon resonance to detect Asian rust in soybean leaf extract at early stages of the disease. The antibody anti-Phakopsora pachyrhizi (pathogen) was covalently immobilized on a gold substrate via a self-assembled monolayer (SAM) of thiols using cysteamine-coupling chemistry. This immunosensor presented a linear response range for the antigen from 3.5 to 28.0 microg mL(-1) (r(2)=0.996). The effects of the antibody amount and the surface blocking to minimize non-specific adsorption on immunosensor response were evaluated. These studies provide new perspectives on using SPR technology for the development of a highly sensitive sensor for agricultural applications.

Short chain regioselectively hydrolyzed scleroglucans induce maturation of porcine dendritic cells.

Branched beta-1,3/1,6-glucans (scleroglucan) were produced by cultivation of Sclerotium rolfsii ATCC 15205. Regioselective hydrolysis at the beta-1,3-linkage of the cell-free and purified polysaccharide was performed in borosilicate glass bottles at pH 5, 121 degrees C, and 1 bar for 72 h. The mixture was divided into four molar mass fractions by stepwise cross-flow filtration using different cutoffs. In vitro studies revealed that scleroglucan hydrolysates with a low molar mass of less than 5 kDa significantly stimulated the activation and maturation of porcine monocyte derived dendritic cells (MoDC) by upregulation of CD40 and CD80/86 as well as by reduction of antigen uptake. MoDC treated with low molar mass scleroglucan showed a considerable increase in the amounts of secreted proinflammatory cytokine tumor necrosis factor alpha and stimulated the proliferation of lymphocytes. Therefore, scleroglucan molecules of low molecular weight are able to induce activation and maturation of porcine DC, which are key initiators of inflammatory and adaptive immune responses, and could provide improved protection against infectious diseases.

Childhood hypersensitivity pneumonitis associated with fungal contamination of indoor hydroponics.

Childhood hypersensitivity pneumonitis (HP) is often associated with exposure to antigens in the home environment. We describe a case of HP associated with indoor hydroponics in a 14-year-old girl. Water samples from hydroponics revealed Aureobasidium pullulans as the dominant fungal micro-organism (10(4)CFU/ml). The diagnosis is supported by the existence of serum precipitating antibodies against A. pullulans, lymphocytic alveolitis on bronchoalveolar lavage (BAL) fluid, a corresponding reaction on a lung biopsy, and the sustained absence of clinical symptoms following the removal of hydroponics from the home. We conclude that hydroponics should be considered as potential sources of fungal contaminants when checking for indoor health complaints.

Anti-tumor and immunomodulating activities of proteoglycans from mycelium of Phellinus nigricans and culture medium.

Two proteoglycans, PNW1 and PNM1, were isolated from the mycelium of Phellinus nigricans through submerged fermentation and culture medium, respectively. PNW1 and PNM1 with similar average molecular weight (33 kDa and 29 kDa) were composed of glucose, galactose, mannose, arabinose and fucose in the molar ratios of 3.26:8.77:6.44:1:1.35 and 20.06:8.72:6.94:1:0.76. At the dose of 100, 200, and 400 mg/kg, PNW1 and PNM1 exhibited anti-tumor activity against mice-transplanted Sarcoma 180 in vivo. However, no direct cytotoxic activity against Sarcoma 180 could be determined. Significant increase in the relative spleen and thymus weight and expression of tumor necrosis factor-alpha (TNF-alpha) in serum was observed, decreasing the tumor weight significantly. PNW1 and PNM1 could stimulate lymphocytes proliferation and increase production of nitric oxide (NO) and TNF-alpha in macrophages. The results indicate that both lymphocyte and macrophages were activated by preparations of proteoglycans from mycelium and culture medium of P. nigricans. The anti-tumor effect of the proteoglycans is not directly tumoricidal but rather immunostimulating.

Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection.

Considerable progress has been made in understanding the function of receptor-like kinase (RLK) genes in model plants. However, much less is known about these genes in crop species. Here we report the characterization of three new wheat RLK genes (TaRLK-R1, 2 and 3). The primary structure of the putative proteins TaRLK-R1, 2 and 3 contained a signal peptide, a cysteine-rich extracellular domain, a transmembrane domain, and a predicted intracellular kinase domain. The fusions between TaRLK-R1, 2 or 3 and the green fluorescence protein (GFP) were targeted to the plasma membrane; such targeting required the signal peptide, extracellular domain and transmembrane domain. Transcription of TaRLK-R1, 2 and 3 was found mainly in the green organs, and was regulated by light. Transcript levels of TaRLK-R1, 2 and 3 increased during the hypersensitive reaction (HR) to stripe rust fungus. In addition, the TaRLK-R3 transcript level was also upregulated by abiotic stresses. Further experiments revealed that the recombinant kinase domain of TaRLK-R3 exhibited auto-phosphorylation activity in vitro. Knocking down the transcript levels of TaRLK-R1, 2 or 3 individually or all together by virus-induced gene silencing compromised the wheat HR to stripe rust fungus. The demonstration of TaRLK-R1, 2 and 3 as positive contributors in the wheat HR to stripe rust fungus suggests a new direction for further functional studies of this important family of RLK genes, and may facilitate the breeding of wheat varieties resistant to stripe rust disease.