Genome-Wide Analysis of Genes Induced by <Emphasis Type="Italic">Fusarium graminearum</Emphasis> Infection in Resistant and Susceptible Wheat Cultivars

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most serious diseases in wheat (Triticum aestivum) and barley (Hordeum vulgare). Dahongmil is an elite Korean wheat cultivar with relatively high resistance to FHB. To identify differentially expressed genes in the resistant cultivar Dahongmil and the susceptible cultivar Urimil after inoculation of F. graminearum, we used the Affymetrix GeneChip® Wheat Genome Array to identify 328 ESTs that were differentially expressed in inoculated seedling tissues of the two cultivars. From these, we selected 16 induced genes and found that they have defense functions, such as genes encoding pathogen resistance proteins, oxidative stress-related proteins, metabolism, and proteins involved in defense mechanisms. To verify the DNA microarray results, we tested seven of these genes by semiquantitative RT-PCR and confirmed that these defense- and stress-related genes were expressed at much higher levels in the resistant Dahongmil cultivar. We next developed a hypothetical functional gene network and identified 89 interaction pairs mediated by four of the differentially expressed genes in the hypothetical network. We further refined the network by identifying nine genes showing significant up- or down-regulation after FHB challenge in the resistant cultivar and two genes having multiple interactions with queried proteins. We hope that the set of induced genes identified in this study can be used for development of new wheat and barley cultivars with improved resistance to FHB.     

Role of hydrogen peroxide and ascorbate-glutathione pathway in host resistance to bacterial wilt of eggplant

Ralstonia solanacearum, a soil-borne bacterium causes bacterial wilt, is a lethal disease of eggplant (Solanum melongena L.). However, the first line of defense mechanism of R. solanacearum infection remains unclear. The present study focused on the role of induced H₂O₂, defense-related enzymes of ascorbate-glutathione pathway variations in resistant and susceptible cultivars of eggplant under biotic stress. Fifteen cultivars of eggplant were screened for bacterial wilt resistance, and the concentration of antioxidant enzymes were estimated upon infection with R. solanacearum. A quantitative real-time PCR was also carried out to study the expression of defense genes. The concentration of H₂O₂ in the pathogen inoculated seedlings was two folds higher at 12 h after pathogen inoculation compared to control. Antioxidant enzymes of ascorbate-glutathione pathway were rapidly increased in resistant cultivars followed by susceptible and highly susceptible cultivars upon pathogen inoculation. The enzyme activity of ascorbate-glutathione pathway correlates by amplification of their defense genes along with pathogenesis-related protein-1a (PR-1a). The expressions of defense genes increased 2.5?3.5 folds in resistant eggplant cultivars after pathogen inoculation. The biochemical and molecular markers provided an insight to understand the first line of defense responses in eggplant cultivars upon inoculation with the pathogen.     

Over-expressing a UDP-glucosyltransferase gene (<Emphasis Type="Italic">Ta-UGT</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript>) enhances <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight resistance of wheat

Wheat Fusarium Head Blight (FHB), mainly caused by Fusarium graminearum (F.g), is a destructive fungal disease worldwide. FHB can not only cause considerable reduction in yield, but more seriously, can contaminate grain by trichothecene toxins released by the fungus. Here, we report new insights into the function and underlying mechanisms of a UDP-glycosyltransferase gene, Ta-UGT ₃ , that is involved in FHB resistance in wheat. In our previous study, Ta-UGT ₃ was found to enhance host tolerance against deoxynivalenol (DON) in Arabidopsis. In this study, four transgenic lines over-expressing Ta-UGT ₃ in a FHB highly susceptible wheat variety, Alondra’s, were obtained and characterized. 3 years of assays using single floret inoculation with F.g indicated that all four transgenic lines exhibited significantly enhanced type II resistance to FHB and less DON accumulation in the grains compared to the untransformed control. Histological observation using GFP labelled F.g was in agreement with the above test results since over-expression of Ta-UGT ₃ dramatically inhibited expansion of F.g. To explore the putative mechanism of resistance mediated by Ta-UGT ₃ , microarray analysis, qRT-PCR and hormone measurements were performed. Microarray analysis showed that DON up-regulated genes, such as TaNPR1, in the susceptible control, and down-regulated genes in F.g inoculated transgenic lines, while qRT-PCR showed that some defence related genes were up-regulated in F.g inoculated transgenic lines. Ta-UGT ₃ over-expression also changed the contents of the endogenous hormones SA and JA in the spikes. These data suggest that Ta-UGT ₃ positively regulates the defence responses to F.g, perhaps by regulating defence-related and DON-induced downstream genes.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

An UDP-Glucosyltransferase Gene from Barley Confers Disease Resistance to <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight

UDP-glucosyltransferases (UGTs) contribute to Fusarium head blight (FHB) resistance of wheat and barley by glycosylating the deoxynivalenol (DON), which is produced by Fusarium fungus. In this study, seven alleles of barley HvUGT14077 (GenBank No.GU170356.1) were cloned using RT-PCR. Among them, HvUGT-10W1, which was isolated from a FHB resistant barley variety 10W1, was significantly up-regulated in young spikes after F. graminearum (F.g) inoculation. HvUGT-10W1::GFP was subcellularly located in the plasma membrane and cytoplasm of the wheat protoplasts. In vitro antifungal activity assay showed that the HvUGT-10W1 protein exerted obvious inhibition against the growth of F.g. The silencing of the HvUGT-10W1 by virus-induced gene silencing (VIGS) resulted in compromised FHB resistance of 10W1, which was shown by the increased infected colonies on the leaves. These indicated that the barley HvUGT-10W1 may also contribute to F.g resistance in barley and provided a potential candidate gene to develop transgenic barley with enhanced FHB resistance.     

Assay development and marker validation for marker assisted selection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> race 1 in watermelon

Fusarium wilt, Fusarium oxysporum f. sp. niveum (FON), of watermelon (Citrullus lanatus) is a fungal pathogen that causes significant yield losses in the US watermelon industry. FON damages watermelon through invasion of the root system and remains a difficult pathogen to manage due to its long-lasting survival spores which persist in the soil. Chemical control options for this pathogen are lacking, making development of genetic resistance the best option. There are four known races of FON (0, 1, 2, and 3) which are distinguished based on their pathogenicity of differential cultivars. Most modern cultivar releases have FON race 1 (FON-1) resistance, which has been mapped on the end of chromosome 1. Application of marker assisted selection (MAS) would improve the efficiency of FON-1 resistance breeding. In order to identify markers for selection in the FON-1 region, the QTL-seq method was utilized on an F₂ population segregating for FON-1 resistance. Single nucleotide polymorphism (SNP) markers in the region were developed into Kompetitive allele-specific PCR (KASP?) assays and tested for trait association on the segregating F_2:3 population. Marker validation was done using an F₂ population from a cross between FON-1 susceptible “New Hampshire Midget” and FON-1-resistant “Calhoun Gray.” Further validation on a panel of susceptible and resistant cultivars and Plant Introductions identified SNP marker UGA1_502161 as a useful marker for selection of FON-1 resistance from Calhoun Gray.     

Biochemical and molecular studies on the resistance mechanisms in tea [<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze] against blister blight disease

Tea (Camellia sinensis) plantations are exposed to biotic and abiotic stresses. Among the biotic factors, blister blight (BB), caused by Exobasidium vexans, affects the quality and quantity of the product and demands high fungicide application. A long term solution for disease resistance would require the knowledge of the basic molecular and biochemical changes occurring in plant as an attempt to resist the pathogen and limit the spread of the disease which can further help in developing resistant cultivars using biotechnological tools. Thus, gene expression studies using the cDNA based suppressive subtractive hybridization library, characterization of genes for pathogenesis related (PR) proteins [chitinase (CsCHIT), glucanase (CsGLUC), phenylalanine ammonia lyase (CsPAL)] and genes in flavonoid pathway were accessed in the BB resistant and susceptible cultivars, SA6 and TES34, respectively. Further, biochemical analysis of PR and antioxidant enzymes (POX, APX, SOD) involved in BB resistance have been carried out to investigate the potential molecular and biochemical changes. Various stages of pathogen development had varied impact on PR protein, flavonoid pathway and anti-oxidative enzymes and indicates the possible role of reactive oxygen species, lignins, flavonoids, anthocyanins and other synthesized compounds in acting as antimicrobial/antifungal agents in tea cultivars.     

FGsub: <Emphasis Type="Italic">Fusarium graminearum</Emphasis> protein subcellular localizations predicted from primary structures

Background

The fungal pathogen Fusarium graminearum (telomorph Gibberella zeae) is the causal agent of several destructive crop diseases, where a set of genes usually work in concert to cause diseases to crops. To function appropriately, the F. graminearum proteins inside one cell should be assigned to different compartments, i.e. subcellular localizations. Therefore, the subcellular localizations of F. graminearum proteins can provide insights into protein functions and pathogenic mechanisms of this destructive pathogen fungus. Unfortunately, there are no subcellular localization information for F. graminearum proteins available now. Computational approaches provide an alternative way to predicting F. graminearum protein subcellular localizations due to the expensive and time-consuming biological experiments in lab.

Results

In this paper, we developed a novel predictor, namely FGsub, to predict F. graminearum protein subcellular localizations from the primary structures. First, a non-redundant fungi data set with subcellular localization annotation is collected from UniProtKB database and used as training set, where the subcellular locations are classified into 10 groups. Subsequently, Support Vector Machine (SVM) is trained on the training set and used to predict F. graminearum protein subcellular localizations for those proteins that do not have significant sequence similarity to those in training set. The performance of SVMs on training set with 10-fold cross-validation demonstrates the efficiency and effectiveness of the proposed method. In addition, for F. graminearum proteins that have significant sequence similarity to those in training set, BLAST is utilized to transfer annotations of homologous proteins to uncharacterized F. graminearum proteins so that the F. graminearum proteins are annotated more comprehensively.

Conclusions

In this work, we present FGsub to predict F. graminearum protein subcellular localizations in a comprehensive manner. We make four fold contributions to this filed. First, we present a new algorithm to cope with imbalance problem that arises in protein subcellular localization prediction, which can solve imbalance problem and avoid false positive results. Second, we design an ensemble classifier which employs feature selection to further improve prediction accuracy. Third, we use BLAST to complement machine learning based methods, which enlarges our prediction coverage. Last and most important, we predict the subcellular localizations of 12786 F. graminearum proteins, which provide insights into protein functions and pathogenic mechanisms of this destructive pathogen fungus.

     

Adult plant stripe rust resistance gene <Emphasis Type="Italic">Yr71</Emphasis> maps close to <Emphasis Type="Italic">Lr24</Emphasis> in chromosome 3D of common wheat

Australian cultivar Sunco carries three adult plant stripe rust resistance genes. One of these genes corresponded to Yr18 in chromosome 7DS; the second, YrCK, was mapped on chromosome 2D. Here, we describe the characterization of the third adult plant resistance (APR) gene from Sunco. Sunco/2*Avocet S-derived lines SA65 (resistant) and SA67 (susceptible) were crossed and a recombinant inbred line F₆ population was generated. Monogenic segregation among SA65/SA67-derived RIL population was demonstrated and the resistance locus was designated YrSA3. Selective genotyping using an iSelect 90 K Infinium SNP array and SSR markers located YrSA3 on chromosome 3D. Development of KASP markers for SNP loci showing association with YrSA3 allowed construction of a genetic map harboring the resistance gene. Ten KASP markers (KASP_8306, KASP_9142, KASP_10438, KASP_16434, KASP_17207, KASP_20836, KASP_23518, KASP_23615, KASP_57983 and KASP_63653), one SSR marker (gwm114b) and Lr24/Sr24 were mapped 1.8 cM distal to YrSA3. Comparison of marker data indicated that the previously named seedling stripe rust resistance gene Yr45 was located proximal to YrSA3, and therefore the latter was formally designated Yr71. Two recombinants carrying Lr24/Sr24 and Yr71 in combination were identified for use as donor sources in wheat breeding programs. The robustness of gwm114b, KASP_16434, KASP_17207 and KASP_20836 for marker-assisted selection of these genes was demonstrated through tests on 74 Australian wheat cultivars.     

Molecular mapping of powdery mildew resistance gene <Emphasis Type="Italic">PmSGD</Emphasis> in Chinese wheat landrace Shangeda using RNA-seq with bulk segregant analysis

Wheat powdery mildew, caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is one of the most devastating diseases of wheat in China and causes serious yield losses. Resistance genes are urgently needed by wheat breeding programs to combat this disease. In the present study, genetic analysis of powdery mildew resistance was conducted on segregated F₂ and F_2:3 populations derived from the cross of Shangeda (providing good resistance to powdery mildew) and Chancellor (susceptible to powdery mildew). The results showed that the resistance of Shangeda to E09 was controlled by a single recessive gene, tentatively designated as PmSGD. In addition, RNA sequencing of the parental lines Shangeda and Chancellor and the corresponding bulked pools derived from homozygous resistant or susceptible F_2:3 lines was implemented to identify single-nucleotide polymorphisms (SNPs). The PmSGD gene was estimated to be located in the 240–250-Mb region of chromosome 7B based on the characteristics of putative SNP loci distributed on 21 wheat chromosomes. Among the developed SNP markers, 17 (57%) markers were linked to PmSGD flanked by SNP2-57 and SNP2-46, with genetic distances of 0.4 and 0.8 cM, respectively. The reaction patterns of Shangeda and cultivars (lines) carrying the Pm5e, Pmhym, mlxbd, and PmTm4 genes to 22 Bgt isolates indicated that PmSGD may be allelic or very closely linked to those genes. All of the SNP loci linked to PmSGD were used to test 38 cultivars with known Pm gene(s), and the results suggested that these SNP loci are useful for pyramiding PmSGD by marker-assisted selection.     

A <Emphasis Type="Italic">bean common mosaic virus</Emphasis> (BCMV)-resistance gene is fine-mapped to the same region as <Emphasis Type="Italic">Rsv1</Emphasis>-<Emphasis Type="Italic">h</Emphasis> in the soybean cultivar Suweon 97

Key message

In the soybean cultivar Suweon 97, BCMV-resistance gene was fine-mapped to a 58.1-kb region co-localizing with the Soybean mosaic virus (SMV)-resistance gene, Rsv1-h raising a possibility that the same gene is utilized against both viral pathogens.

Abstract

Certain soybean cultivars exhibit resistance against soybean mosaic virus (SMV) or bean common mosaic virus (BCMV). Although several SMV-resistance loci have been reported, the understanding of the mechanism underlying BCMV resistance in soybean is limited. Here, by crossing a resistant cultivar Suweon 97 with a susceptible cultivar Williams 82 and inoculating 220 F₂ individuals with a BCMV strain (HZZB011), we observed a 3:1 (resistant/susceptible) segregation ratio, suggesting that Suweon 97 possesses a single dominant resistance gene against BCMV. By performing bulked segregant analysis with 186 polymorphic simple sequence repeat (SSR) markers across the genome, the resistance gene was determined to be linked with marker BARSOYSSR_13_1109. Examining the genotypes of nearby SSR markers on all 220 F₂ individuals then narrowed down the gene between markers BARSOYSSR_13_1109 and BARSOYSSR_13_1122. Furthermore, 14 previously established F_2:3 lines showing crossovers between the two markers were assayed for their phenotypes upon BCMV inoculation. By developing six more SNP (single nucleotide polymorphism) markers, the resistance gene was finally delimited to a 58.1-kb interval flanked by BARSOYSSR_13_1114 and SNP-49. Five genes were annotated in this interval of the Williams 82 genome, including a characteristic coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR, CNL)-type of resistance gene, Glyma13g184800. Coincidentally, the SMV-resistance allele Rsv1-h was previously mapped to almost the same region, thereby suggesting that soybean Suweon 97 likely relies on the same CNL-type R gene to resist both viral pathogens.

     

Genetic analysis and location of a resistance gene for <Emphasis Type="BoldItalic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="BoldItalic">tritici</Emphasis> in wheat cultivar Zhengmai 7698

Wheat stripe (yellow) rust, caused by Puccinia striiformis West. f. sp. tritici (Pst), is one of the most destructive diseases in many wheat-growing countries, especially in China, the largest stripe rust epidemic area in the world. Growing the resistant cultivars is an effective, economic and environmentally friendly way to control this disease. Wheat cultivar Zhengmai 7698 has shown a high-level resistance to wheat stripe rust. To elucidate its genetic characteristics and location of the resistance gene, Zhengmai 7698 was crossed with susceptible variety Taichung 29 to produce \(\hbox {F}_{{1}}\), \(\hbox {F}_{{2}}\) and \(\hbox {BC}_{{1}}\) progeny generations. The genetic analysis showed that the stripe rust resistance in Zhengmai 7698 to Pst predominant race CYR32 was controlled by a single-dominant gene, namedYrZM. Bulked segregant analysis and simple sequence repeat (SSR) markers were used to map the gene. Four SSR markers, Xbarc198, Xwmc179, Xwmc786 and Xwmc398 on chromosome 6BL were polymorphic between the parents and resistance, and susceptible bulks. A linkage genetic map was constructed using 212 \(\hbox {F}_{{2}}\) plants in the sequential order of Xwmc398, Xwmc179, YrZM, Xbarc198, Xwmc786. As this gene is effective against predominant race CYR32, it is useful in combination with other resistance genes for developing new wheat cultivars with resistance to stripe rust.     

Differential fungal colonization and physiological defense responses of new olive cultivars infected by the necrotrophic fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

New olive cultivars adapted to Tunisia’s growing conditions were examined for their resistance to verticillium wilt (VWO) to determine whether differences in susceptibility among currently grown cultivars might contribute to the management of this disease. Based on the evaluation of 14 cultivars, 10 were classified as susceptible or extremely susceptible (Chetoui, Chemlali, Rkhami, Jarboui, Zalmati, Jarboui, Oueslati, Manzanille, Picholine and Frangivento), 2 as moderately susceptible (Koroneiki and Coratina), and 2 as resistant (Meski and Sayali) to VWO. Three cultivars with different susceptibility levels were selected to examine the levels of hydrogen peroxide (H₂O₂), soluble sugars (SS), soluble proteins (SP), total polyphenols (TP), lipid peroxidation, activities of antioxidant enzymes, and fungal biomass in planta. V. dahliae DNA occurred early in the roots at 15 dpi and reached a maximum of 3.507 and 2.52 ng/100 ng of plant DNA, respectively, in the extremely susceptible and resistant cultivars. Fungal DNA in the stems occurred at 30 dpi and increased slowly to reach a maximum of 0.23 ng/100 ng of total DNA in the extremely susceptible cultivars. We showed that the amount of fungal DNA in planta was roughly correlated with the susceptibility to VWO (P < 0.0001; r = 0.95). The comparison of cultivars at the physiological level indicated that olive resistance is roughly correlated with the antioxidant enzymes activity, H₂O₂ concentration, and TP and SP contents. The results of this study open new perspectives for olive genetic improvement programs aiming at developing new cultivars resistant to this wilt.     

Characterization and evaluation of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strain WF02 regarding its biocontrol activities and genetic responses against bacterial wilt in two different resistant tomato cultivars

Bacillus amyloliquefaciens strain WF02, isolated from soil collected at Wufeng Mountain, Taiwan, has siderophore-producing ability and in vitro antagonistic activity against bacterial wilt pathogen. To determine the impact of plant genotype on biocontrol effectiveness, we treated soil with this strain before infecting susceptible (L390) and moderately resistant (Micro-Tom) tomato cultivars with Ralstonia solanacearum strain Pss4. We also compared the efficacy of this strain with that of commercial Bacillus subtilis strain Y1336. Strain WF02 provided longer lasting protection against R. solanacearum than did strain Y1336 and controlled the development of wilt in both cultivars. To elucidate the genetic responses in these plants under WF02 treatment, we analyzed the temporal expression of defense-related genes in leaves. The salicylic acid pathway-related genes phenylalanine ammonia-lyase and pathogenesis-related protein 1a were up-regulated in both cultivars, whereas expression of the jasmonic acid pathway-related gene lipoxygenase was only elevated in the susceptible tomato cultivar (L390). These results suggest that WF02 can provide protection against bacterial wilt in tomato cultivars with different levels of disease resistance via direct and indirect modes of action.     

Time-course of pathogen induced accumulation of callose as mechanical protective barrier in wheat seedlings

Results of fluorescence microscopic study and quantitative luminescent analysis of pathogen induced callose accumulation in winter wheat seedlings of two cultivars with different resistance to causal agent of eyespot disease are presented. Higher content of constitutive callose in intact seedlings of unsusceptible cultivar at the initial stages of vegetation was determined. It correlates with resistance of this cultivar to Pseudocercosporella herpotrichoides—causal agent of eyespot disease. The increased pathogen induced accumulation of callose in seedlings of susceptible cultivar was revealed, however, it did not influence it protection against pathogen.     

Identifying molecular markers suitable for <Emphasis Type="Italic">Frl</Emphasis> selection in tomato breeding

Modern plant breeding heavily relies on the use of molecular markers. In recent years, next generation sequencing (NGS) emerged as a powerful technology to discover DNA sequence polymorphisms and generate molecular markers very rapidly and cost effectively, accelerating the plant breeding programmes. A single dominant locus, Frl, in tomato provides resistance to the fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL), causative agent of Fusarium crown and root rot. In this study, we describe the generation of molecular markers associated with the Frl locus. An F₂ mapping population between an FORL resistant and a susceptible cultivar was generated. NGS technology was then used to sequence the genomes of a susceptible and a resistant parent as well the genomes of bulked resistant and susceptible F₂ lines. We zoomed into the Frl locus and mapped the locus to a 900 kb interval on chromosome 9. Polymorphic single-nucleotide polymorphisms (SNPs) within the interval were identified and markers co-segregating with the resistant phenotype were generated. Some of these markers were tested successfully with commercial tomato varieties indicating that they can be used for marker-assisted selection in large-scale breeding programmes.