A matter of life and death: The power of personal networks for medical crowdfunding performance.

Medical crowdfunding is an accessible alternative for individuals to meet their unaffordable health needs. This study explores the role of personal networks in medical crowdfunding performance from the perspective of tie strength and whether gender inequality persists in the returns of personal networks in this survival context, using bilateral data of both the ego and the alters collected from a large representative medical crowdfunding platform in China. It is found that kin ties play a fundamental and predominant role while pseudo-kin ties, being less strong than kin ties in terms of mutual sentiment and reciprocal obligations to help each other, play an accumulative role and are more influential in increasing crowdfunding performance, and neighbour and other role relations have the weakest effect and contribution. Importantly, women are not discriminated against when mobilizing personal networks for medical crowdfunding as they enjoy the same returns of most personal ties as men do.

Genome-wide identification of glutamate receptor genes in adzuki bean and the roles of these genes in light and rust fungal response.

Plant glutamate receptor proteins (GLRs) are involved in plant development, biotic stress, and light-signal transduction. Vigna angularis is a traditional crop with important economic value in China, and the identification of functional genes can facilitate the breeding of stress resistant varieties. Here, we identified the members of the GLR gene family in the adzuki bean genome and investigated gene expression under light and rust fungal (Uromyces vignae) stimuli. Sixteen GLR genes were identified in V. angularis (VaGLRs), and these genes clustered in a single clade (clade III) with two groups. Evolutionary analysis showed that three VaGLRs result from tandem duplications and four result from whole genome/segmental duplications. To understand the regulation of expression of VaGLRs, cis-acting elements were analyzed in the promoter regions of the VaGLRs including cis-acting elements associated with light and stress responsiveness. Expression analysis by qRT-PCR revealed transcripts of eight and 10 VaGLRs in response to light stimuli and rust infection, respectively. For light responsiveness, the expression levels of XP_017430569.1 and XP_017425299.1 were higher under light condition than in darkness, while the expression levels of XP_017406996.1, XP_017425763.1, and XP_017423557.1 gradually recovered during dark treatment. Additionally, the relative expression levels of XP_017413816.1, XP_017436268.1, and XP_017425299.1 were significantly elevated during U. vignae infection in a resistant cultivar compared to the expression levels in a susceptible cultivar. XP_017425299.1 expression was induced both by light and rust infection, suggesting this gene may link light and disease resistance signaling pathways. Our results provide insight into how the VaGLRs contribute to adzuki bean response to light stimulus and pathogen attack. These identified VaGLRs also provide important reference to improve adzuki bean germplasm resources.

A natriuretic peptide molecule from Vigna angularis, VaEG45, confers rust resistance by inhibiting fungal development.

KEY MESSAGE: Novel function and mechanism of a PNP molecule VaEG45 from adzuki bean involved in plant immunity. Plant natriuretic peptides (PNPs) can affect a broad spectrum of physiological responses in plants acting as peptidic signaling molecules. However, PNPs may play additional roles in plant immunity. Our previous transcriptome data of adzuki bean (Vigna angularis) in response to Uromyces vignae infection revealed association of PNP-encoding gene VaEG45 with U. vignae resistance. To determine the function of VaEG45 in disease resistance, we cloned the 589 bp nucleotide sequence of VaEG45 containing 2 introns, encoding a putative 13.68 kDa protein that is 131 amino acids in length. We analyzed expression in different resistant cultivars of V. angularis and found significant induction of VaEG45 expression after U. vignae infection. Transient expression of VaEG45 improved tobacco resistance against Botrytis cinerea. We next analyzed the mechanism by which VaEG45 protects plants from fungal infection by determination of the biological activity of the prokaryotic expressed VaEG45. The results showed that the fusion protein VaEG45 can significantly inhibit urediospores germination of U. vignae, mycelial growth, and the infection of tobacco by B. cinerea. Further analysis revealed that VaEG45 exhibits ß-1, 3-glucanase activity. These findings uncover the function of a novel PNP molecule VaEG45 and provide new evidence about the mechanism of PNPs in plant immunity.

Histological and molecular responses of Vigna angularis to Uromyces vignae infection.

BACKGROUND: To advance the understanding of adzuki bean (Vigna angularis) resistance to infection with the rust-causing fungus Uromyces vignae (Uv), we comprehensively analyzed histological events and the transcriptome of Uv-infected adzuki bean. RESULTS: Compared with the susceptible cv. Baoqinghong (BQH), the resistant cv. QH1 showed inhibition of uredospore germination and substomatal vesicle development, intense autofluorescence of cells around the infection site, and cell wall deposit formation in response to Uv infection. In cv. QH1, gene set enrichment analysis (GSEA) showed enrichment of chitin catabolic processes and responses to biotic stimuli at 24 h post-inoculation (hpi) and cell wall modification and structural constituent of cytoskeleton at 48 hpi. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated enrichment of WRKY transcription factors (TFs), the calcium binding protein cml, and hydroquinone glucosyltransferase at both 24 and 48 hpi. In total, 1992 and 557 differentially expressed genes (DEGs) were identified at 24 and 48 hpi, respectively. Cell surface pattern-recognition receptors (PRRs), WRKY TFs, defense-associated pathogenesis-related (PR) proteins, and lignin and antimicrobial phenolic compound biosynthesis were significantly induced. Finally, we detected the chitinase (CHI) and phenylalanine ammonia-lyase (PAL) activity were higher in QH1 and increased much earlier than in BQH. CONCLUSION: In cv. QH1, cell-surface PRRs rapidly recognize Uv invasion and activate the corresponding TFs to increase the transcription of defense-related genes and corresponding enzymatic activities to prevent fungal development and spread in host tissues.

Unconventional Recombination in the Mating Type Locus of Heterothallic Apple Canker Pathogen Valsa mali.

Sexual reproduction in filamentous ascomycetes is controlled by the mating type (MAT) locus, including two idiomorphs MAT1-1 and MAT1-2 Understanding the MAT locus can provide clues for unveiling the sexual development and virulence factors for fungal pathogens. The genus Valsa (Sordariomycetes, Diaporthales) contains many tree pathogens responsible for destructive canker diseases. The sexual stage of these ascomycetes is occasionally observed in nature, and no MAT locus has been reported to date. Here, we identified the MAT locus of the apple canker pathogen Valsa mali, which causes extensive damage, and even death, to trees. V. mali is heterothallic in that each isolate carries either the MAT1-1 or MAT1-2 idiomorph. However, the MAT structure is distinct from that of many other heterothallic fungi in the Sordariomycetes. Two flanking genes, COX13 and APN2, were coopted into the MAT locus, possibly by intrachromosomal rearrangement. After the acquisition of foreign genes, unequal recombination occurred between MAT1-1/2 idiomorphs, resulting in a reverse insertion in the MAT1-2 idiomorph. Evolutionary analysis showed that the three complete MAT1-1-2, COX13, and APN2 genes in this region diverged independently due to different selection pressure. Null hypothesis tests of a 1:1 MAT ratio of 86 V. mali isolates from four different provinces showed a relatively balanced distribution of the two idiomorphs in the fields. These results provide insights into the evolution of the mating systems in Sordariomycetes.

Selection and Validation of Reference Genes for Gene Expression Analysis in Vigna angularis Using Quantitative Real-Time RT-PCR.

Adzuki bean (Vigna angularis) is one of the most important legume crops in Asian countries like China, Japan and Korea due to its nutritious protein and starch contents. In spite of its economic importance, gene expression analysis system for gene function verification of adzuki bean is still absent. Therefore, reference genes for gene expression analysis based on the quantitative real time PCR (qRT-PCR) were screened in current study. A total of nine general housekeeping genes, including ACT, Fbox, ZMPP, GAPDH, EF, PP2A, UBC, UBN and PTB were evaluated for their expression stability by qRT-PCR in four adzuki bean cultivars, three different tissues, four abiotic stress and one biotic stress. The best group of candidates as reference genes were as follows: PTB and ACT for different cultivars; EF and UBN for different tissues; ACT and ZMPP for biotic stress and waterlogging stress; Fbox and UBC for salinity-alkalinity stress; Fbox and PTB for drought stress. Our results will provide a more accurate and reliable normalization of qRT-PCR data in adzuki bean.

Clg2p interacts with Clf and ClUrase to regulate appressorium formation, pathogenicity and conidial morphology in Curvularia lunata.

Ras is a small GTPase that regulates numerous processes in the cellular development and morphogenesis of many organisms. In this study, we identified and functionally characterized the Clg2p gene of Curvularia lunata, which is homologous with the Ras protein. The Clg2p deletion mutant (ΔClg2p) had altered appressorium formation and conidial morphology and produced fewer, smaller lesions compared with the wild-type strain. When a dominant Clg2p allele was introduced into the mutant, all of these defective phenotypes were completely restored. To further understand the regulation of Clg2p in appressorium formation and conidial morphology, and its role in pathogenicity, seven Clg2p-interacting proteins were screened using a yeast two-hybrid assay. Two of these proteins, Clf, a homologue of Mst11, which corresponds to MAP kinase kinase kinase in Magnaporthe oryzae, and urate oxidase (designated ClUrase) were functionally characterized. Clg2p specifically interacted with Clf through its RA domain to regulate appressorium formation and pathogenicity, whereas the Clg2p-ClUrase interaction regulated conidial morphology without affecting fungal pathogenicity. This report is the first to elucidate the regulatory mechanism of the key Ras protein Clg2p in C. lunata.

Studies on the Infection, Colonization, and Movement of Pseudomonas syringae pv. actinidiae in Kiwifruit Tissues Using a GFPuv-Labeled Strain.

Kiwifruit bacterial canker, an economically important disease caused by Pseudomonas syringae pv. actinidiae (Psa), has caused severe losses in all major areas of kiwifruit cultivation. Using a GFPuv-labeled strain of Psa, we monitored the invasion, colonization, and movement of the pathogen in kiwifruit twigs, leaves and veins. The pathogen can invade twigs through both wounds and natural openings; the highest number of Psa is obtained in cut tissues. We determined that, following spray inoculation, Psa-GFPuv could infect leaves and cause lesions in the presence and absence of wounds. Light and transmission electron microscopic observations showed that bacterial cells colonize both phloem and xylem vessels. Bacterial infection resulted in marked alterations of host tissues including the disintegration of organelles and degeneration of protoplasts and cell walls. Furthermore, low temperature was conducive to colonization and movement of Psa-GFPuv in kiwifruit tissues. Indeed, the pathogen migrated faster at 4°C than at 16°C or 25°C in twigs. However, the optimum temperature for colonization and movement of Psa in leaf veins was 16°C. Our results, revealing a better understanding of the Psa infection process, might contribute to develop more efficacious disease management strategies.

Genome sequence of Valsa canker pathogens uncovers a potential adaptation of colonization of woody bark.

Canker caused by ascomycetous Valsa species are among the most destructive diseases of woody plants worldwide. These pathogens are distinct from other pathogens because they only effectively attack tree bark in the field. To unravel the potential adaptation mechanism of bark colonization, we examined the genomes of Valsa mali and Valsa pyri that preferentially infect apple and pear, respectively. We reported the 44.7 and 35.7 Mb genomes of V. mali and V. pyri, respectively. We also identified the potential genomic determinants of wood colonization by comparing them with related cereal pathogens. Both genomes encode a plethora of pathogenicity-related genes involved in plant cell wall degradation and secondary metabolite biosynthesis. In order to adapt to the nutrient limitation and low pH environment in bark, they seem to employ membrane transporters associated with nitrogen uptake and secrete proteases predominantly with acidic pH optima. Remarkably, both Valsa genomes are especially suited for pectin decomposition, but are limited in lignocellulose and cutin degradation. Besides many similarities, the two genomes show distinct variations in many secondary metabolism gene clusters. Our results show a potential adaptation of Valsa canker pathogens to colonize woody bark. Secondary metabolism gene clusters are probably responsible for this host specificity.

[Identification of a fungal isolate causing adzuki bean rust in Heilongjiang].

OBJECTIVE: The aim of the study is to identify the pathogen causing adzuki bean (Phaseolus angularis) rust in Daqing, Heilongjiang province. METHODS: Adzuki bean rust leaves were collected from Daqing, Heilongjiang province, China. A pure culture of rust isolate ZXL01 was obtained by single pustule isolation. Its taxonomic status was determined by observing the number of germ pores of urediniospores, germ pore location and the wall thickness of teliopores, and sequencing ribosomal DNA internal transcribed spacer (rDNA-ITS). RESULTS: Morphological studies showed that most of the urediospores of ZXL01 had two germ pores that were far from spores' equator area. The wall thickness of teliopores ranged from 2.9 to 3. 3 microm. The rDNA-ITS sequence of ZXL01 was clustered in one clade with 2 reference isolates of Uromyces vignae (GenBank accession numbers AB115718 and AB115731) at 99% bootstrap levels in the phylogenetic tree. A 500 bp amplified product was obtained by the specific primers UV-ITSF/R, which was specific for U. Vignae. CONCLUSION: The morphological features and ITS analysis indicated that the rust fungus ZXL01 occurred on leaves of adzuki bean in Daqing was U. Vignae, and the accession number of GenBank was KM461700.

Transcriptome profiling to identify genes involved in pathogenicity of Valsa mali on apple tree.

Apple Valsa canker, caused by the fungus Valsa mali (Vm), is one of the most destructive diseases of apple in China. A better understanding of this host-pathogen interaction is urgently needed to improve management strategies. In the current study we sequenced the transcriptomes of Vm during infection of apple bark and mycelium grown in axenic culture using Illumina RNA-Seq technology. We identified 437 genes that were differentially expressed during fungal infection compared to fungal mycelium grown in axenic culture. One hundred and thirty nine of these 437 genes showed more than two fold higher transcript abundance during infection. GO and KEGG enrichment analyses of the up-regulated genes suggest prevalence of genes associated with pectin catabolic, hydrolase activity and secondary metabolite biosynthesis during fungal infection. Some of the up-regulated genes associated with loss of pathogenicity and reduced virulence annotated by host-pathogen interaction databases may also be involved in cell wall hydrolysis and secondary metabolite transport, including a glycoside hydrolase family 28 protein, a peptidase and two major facilitator superfamily proteins. This highlights the importance of secondary metabolites and cell wall hydrolases during establishment of apple Valsa canker. Functional verification of the genes involved in pathogenicity of Vm will allow us to better understand how the fungus interferes with the host machinery and assists in apple canker establishment.

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR.

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.

Exogenous melatonin improves Malus resistance to Marssonina apple blotch.

We examined whether exogenously applied melatonin could improve resistance to Marssonina apple blotch (Diplocarpon mali) by apple [Malus prunifolia (Willd.) Borkh. cv. Donghongguo]. This serious disease leads to premature defoliation in the main regions of apple production. When plants were pretreated with melatonin, resistance was increased in the leaves. We investigated the potential roles for melatonin in modulating levels of hydrogen peroxide (H2O2), as well the activities of antioxidant enzymes and pathogenesis-related proteins during these plant-pathogen interactions. Pretreatment enabled plants to maintain intracellular H2O2 concentrations at steady-state levels and enhance the activities of plant defence-related enzymes, possibly improving disease resistance. Because melatonin is safe and beneficial to animals and humans, exogenous pretreatment might represent a promising cultivation strategy to protect plants against this pathogen infection.

A Nested PCR Assay for Detecting Valsa mali var. mali in Different Tissues of Apple Trees.

A nested polymerase chain reaction (PCR) assay for detecting Valsa mali var. mali, the causal agent of apple tree Valsa canker, was developed. One pair of genus-specific primers was designed based on the ribosomal DNA internal transcribed spacer conservative sequence of the Valsa genus and one pair of species-specific primers was designed based on the specific sequence of V. mali var. mali. The specificity of the genus-specific and species-specific primers was evaluated against 10 V. mali var. mali isolates, 10 V. mali var. pyri isolates, 4 isolates from closely related Valsa spp., and 8 isolates from fungal species that are commonly isolated from naturally infected apple bark tissue. A distinct band of 348 bp in length was detected in all V. mali var. mali isolates but not in other tested species and the V. mali var. pyri variety. The sensitivity of this assay was evaluated by serial dilutions of DNA extracted from V. mali var. mali pure cultures and apple bark tissues with or without visible symptoms. The results showed that the assay was able to detect as little as 100 fg of DNA in mycelial samples and apple bark tissues with visible symptoms, whereas the lowest detectable concentration was 10 pg of DNA in symptomless apple bark tissues. The efficiency of the nested PCR assay was compared with that of fungal isolation assays. All symptomless and symptomatic samples from which the pathogen was successfully isolated yielded a PCR product of the expected size. The detection rate of nested PCR for symptomless samples was 64.7%, which was much higher than the detection rate of 20.6% by fungal isolation. The PCR analysis of different symptomless tissues showed that the incidence of V. mali var. mali was different in different tissues of apple trees. The average incidence of V. mali var. mali was 89% in terminal buds, 71% in internodes, and 48% in bud scale scars. Moreover, the incidence of V. mali var. mali in nonsymptomatic tissues was higher in orchards where more trees were infected. Taken together, the assay developed in this study can be used for rapid and reliable detection of V. mali var. mali in tissues of apple trees with or without symptoms and also for monitoring the presence of the pathogen at an early stage of disease development.

[Development of genetic transformation system of Valsa mali of apple mediated by PEG].

OBJECTIVE: The genetic transformation of Valsa mali var. mali was developed by PEG-mediated protoplasts transformation. METHOD: It was transformed by PEG-induced fusion of protoplasts. The plasmid pBIG2RHPH2-GFP-GUS carrying hph gene was used and Valsa mali var. mali 03-8 isolate was used as the host strain. RESULT: At 50 mg/mL driselase + 10 mg/mL lysing enzymes concentration, the mycelium of Valsa mali var. mali cultured in YEPD medium for 48 h was hydrolyzed in 10 mL enzymes liquid /0.5 g wet mycelium for 2 h. The protoplast yield was 4 x 10(7) CFU/mg. The transformation efficiency was 44 per g DNA. Analysis of the transformants by PCR and Southern blotting showed that the selectable marker gene hph was integrated effectively into the genome of Valsa mali var. mali. After 5 subculturing on PDA, 87.5% transformants could grow. This stability test of transformants suggested that the foreign gene hph was stable in heredity. CONCLUSION: This transformation system is a valuable and important tool for the further study of the pathogenic gene of Valsa mali.