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SummaryOverexpressors ofGlycine max disease resistant 1(GmDR1) exhibits broad-spectrum resistance againstFusarium virguliforme, soybean cyst nematode (SCN), spider mites, and soybean aphids in soybean. To understand the mechanisms of broad-spectrum immunity mediated byGmDR1, we investigated the transcriptomes of a strong and a weakGmDR1-overexpressors following treatment with chitin, a pathogen- and pest-associated molecular pattern (PAMP) found in these organisms. The strong and weakGmDR1-overexpressors exhibited altered expression of 6,098 and 992 genes, respectively, as compared to the nontransgenic control following chitin treatment. However, only 192 of these genes exhibited over two-fold changes in expression levels in both strong and weakGmDR1-overexpressors as compared to the control. MapMan analysis of the 192 genes revealed 64 biotic stress-related genes, of which 53 were induced and 11 repressed as compared to the control. The 53 chitin-induced genes include nine that encode receptor kinases, 13 encodenucleotide-binding leucine-richrepeat (NLR) receptor proteins, seven encoding WRKY transcription factors, four ethylene response factors, and three MYB-like transcription factors. Investigation of a subset of these genes revealed three receptor protein kinases, seven NLR proteins, and one WRKY transcription factor genes that are induced followingF. virguliformeand SCN infection. The integral plasma membrane GmDR1 protein most likely recognizes PAMPs including chitin and activates transcription of genes encoding receptor kinases and NLR proteins. GmDR1 could be a pattern recognition receptor that regulates the expression of several NLRs for expression of PAMP-triggered immunity and/or priming the effector triggered immunity.

The Phytophtora root and stem rot is a serious disease in soybean. It is caused by the oomycete pathogen Phytophthora sojae. Growing Phytophthora resistant cultivars is the major method of controlling this disease. Resistance is race- or gene-specific; a single gene confers immunity against only a subset of the P. sojae isolates. Unfortunately, rapid evolution of new Phytophthora sojae virulent pathotypes limits the effectiveness of an Rps (“resistance to Phytophthora sojae”) gene to 8–15 years. The current study was designed to investigate the effectiveness of Rps12 against a set of P. sojae isolates using recombinant inbred lines (RILs) that contain recombination break points in the Rps12 region. Our study revealed a unique Rps gene linked to the Rps12 locus. We named this novel gene as Rps13 that confers resistance against P. sojae isolate V13, which is virulent to recombinants that contains Rps12 but lack Rps13. The genetic distance between the two Rps genes is 4 cM. Our study revealed that two tightly linked functional Rps genes with distinct race-specificity provide broad-spectrum resistance in soybean. We report here the molecular markers for incorporating the broad-spectrum Phytophthora resistance conferred by the two Rps genes in commercial soybean cultivars.

International audience; Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 2013), the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani Species Complex (FSSC). Subsequently, this concept was challenged by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). Here we test this claim, and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species recently described as Neocosmospora were recombined in Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural and practical taxonomic option available.

Summary Plants fight‐off pathogens and pests by manifesting an array of defence responses using their innate immunity mechanisms. Here we report the identification of a novel soybean gene encoding a plasma membrane protein, transcription of which is suppressed following infection with the fungal pathogen, Fusarium virguliforme. Overexpression of the protein led to enhanced resistance against not only against F. virguliforme, but also against spider mites (Tetranychus urticae, Koch), soybean aphids (Aphis glycines, Matsumura) and soybean cyst nematode (Heterodera glycines). We, therefore, name this protein as Glycine max disease resistance 1 (GmDR1; Glyma.10g094800). The homologues of GmDR1 have been detected only in legumes, cocoa, jute and cotton. The deduced GmDR1 protein contains 73 amino acids. GmDR1 is predicted to contain an ecto‐ and two transmembrane domains. Transient expression of the green fluorescent protein fused GmDR1 protein in soybean leaves showed that it is a plasma membrane protein. We investigated if chitin, a pathogen‐associated molecular pattern (PAMP), common to all pathogen and pests considered in this study, can significantly enhance defence pathways among the GmDR1‐overexpressed transgenic soybean lines. Chitin induces marker genes of the salicylic‐ and jasmonic acid‐mediated defence pathways, but suppresses the defence pathway regulated by ethylene. Chitin induced SA‐ and JA‐regulated defence pathways may be one of the mechanisms involved in generating broad‐spectrum resistance among the GmDR1‐overexpressed transgenic soybean lines against two serious pathogens and two pests including spider mites, against which no known resistance genes have been identified in soybean and among the most other crop species.

Cold stress adversely affects the growth and development of plants and limits the geographical distribution of crop plants. Accumulation of spontaneous mutations shapes the adaptation of plant species to diverse climatic conditions. Genome-wide association study of the phenotypic variation gathered by a newly designed phenomic platform with that of the single nucleotide polymorphic (SNP) loci across the genomes of 417 Arabidopsis natural variants collected from various geographical regions revealed 33 candidate genes for cold tolerance. Investigation of at least two independent mutants for 29 of these genes identified 16 cold tolerance genes controlling diverse genetic mechanisms. This study identified five genes encoding novel leucine-rich repeat domain-containing proteins, including three nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. Among the 16 identified cold tolerance genes, ADS2 and ACD6 are the only two cold tolerance genes identified earlier. The comparatively little overlap between the genes identified in this genome-wide association study of natural variants with those discovered previously through forward and reverse genetic approaches suggests that cold tolerance is a complex physiological process governed by a large number of genetic mechanisms.Short SummaryCold stress adversely affects the growth and development of plants and limits the geographical distribution of crop plants. Genome-wide association study of the phenotypic variation of Arabidopsis natural variants with that of the single nucleotide polymorphic loci followed by T-DNA insertion mutant analyses of 29 candidate genes led to assigning cold tolerance function for the first time to 14 genes including three nucleotide-binding sites leucine repeat region (NB-LRR) protein genes. The comparatively little overlap between the genes identified in this study with those discovered previously suggests that cold tolerance is governed by a complex network of multiple genetic mechanisms.

Phytophthora sojae is an oomycete that causes stem and root rot disease in soybean. P. sojae delivers many RxLR effector proteins, including Avr1b, into host cells to promote infection. We show here that Avr1b interacts with the soybean U-box protein, GmPUB1-1, in yeast two-hybrid, pull down, and bimolecular fluorescence complementation (BIFC) assays. GmPUB1-1, and a homeologous copy GmPUB1-2, are induced by infection and encode 403 amino acid proteins with U-Box domains at their N-termini. Non-synonymous mutations in the Avr1b C-terminus that abolish suppression of cell death also abolished the interaction of Avr1b with GmPUB1-1, while deletion of the GmPUB1-1 C-terminus, but not the U box, abolished the interaction. BIFC experiments suggested that the GmPUB1-1-Avr1b complex is targeted to the nucleus. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the GmPUB1 genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by Rps1-b and Rps1-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by Rps1-k plants was not, however, affected following GmPUB1-1 silencing. Furthermore, over-expression of GmPUB1-1 in particle bombardment experiments triggered cell death suggesting that GmPUB1 may be a positive regulator of effector-triggered immunity. In a yeast two-hybrid system, GmPUB1-1 also interacted with a number of other RxLR effectors including Avr1d, while Avr1b and Avr1d interacted with a number of other infection-induced GmPUB proteins, suggesting that the pathogen uses a multiplex of interactions of RxLR effectors with GmPUB proteins to modulate host immunity.

Phytophthora sojae is an oomycete that causes stem and root rot disease in soybean. P. sojae delivers many RxLR effector proteins, including Avr1b, into host cells to promote infection. We show here that Avr1b interacts with the soybean U-box protein, GmPUB1-1, in yeast two-hybrid, pull down, and bimolecular fluorescence complementation (BIFC) assays. GmPUB1-1, and a homeologous copy GmPUB1-2, are induced by infection and encode 403 amino acid proteins with U-Box domains at their N-termini. Non-synonymous mutations in the Avr1b C-terminus that abolish suppression of cell death also abolished the interaction of Avr1b with GmPUB1-1, while deletion of the GmPUB1-1 C-terminus, but not the U box, abolished the interaction. BIFC experiments suggested that the GmPUB1-1-Avr1b complex is targeted to the nucleus. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the GmPUB1 genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by Rps1-b and Rps1-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by Rps1-k plants was not, however, affected following GmPUB1-1 silencing. Furthermore, over-expression of GmPUB1-1 in particle bombardment experiments triggered cell death suggesting that GmPUB1 may be a positive regulator of effector-triggered immunity. In a yeast two-hybrid system, GmPUB1-1 also interacted with a number of other RxLR effectors including Avr1d, while Avr1b and Avr1d interacted with a number of other infection-induced GmPUB proteins, suggesting that the pathogen uses a multiplex of interactions of RxLR effectors with GmPUB proteins to modulate host immunity.

Nonhost resistance (NHR), which protects all members of a plant species from non-adapted or nonhost plant pathogens, is the most common form of plant immunity. NHR provides the most durable and robust form of broad-spectrum immunity against non-adaptive pathogens pathogenic to other crop species. In a mutant screen for loss of Arabidopsis (Arabidopsis thaliana) NHR against the soybean pathogen, Phytophthora sojae, the Phytophthora sojae susceptible 30 (pss30) mutant was identified. The pss30 mutant is also susceptible to the soybean pathogen Fusarium virguliforme. PSS30 encodes a folate transporter, AtFOLT1, which was previously localized to chloroplasts and implicated in the transport of folate from the cytosol to plastids. We have shown that two Arabidopsis folate biosynthetic mutants with reduced folate levels exhibited a loss of nonhost immunity against P. sojae. As compared to the wild-type Col-0 ecotype, the steady state folate levels are reduced in the pss1, atfolt1 and two folate biosynthetic mutants suggesting that folate is required for expression of nonhost immunity. Overexpression of AtFOLT1 enhances immunity of transgenic soybean (Glycine max, (L.) Merr.) lines against two serious soybean pathogens, the fungal pathogen F. virguliforme and soybean cyst nematode (SCN) Heterodera glycines. Transgenic lines showing enhanced SCN resistance also showed a trend of increased levels of folate accumulation. This study thus suggests that folate contributes to nonhost plant immunity and overexpression of a nonhost resistance gene could be a suitable strategy for generating broad-spectrum disease resistance in crop plants.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the

Novel QTL conferring resistance to both the SDS and SCN was detected in two RIL populations. Dual resistant RILs could be used in breeding programs for developing resistant soybean cultivars. Soybean cultivars, susceptible to the fungus Fusarium virguliforme, which causes sudden death syndrome (SDS), and to the soybean cyst nematode (SCN) (Heterodera glycines), suffer yield losses valued over a billion dollars annually. Both pathogens may occur in the same production fields. Planting of cultivars genetically resistant to both pathogens is considered one of the most effective means to control the two pathogens. The objective of the study was to map quantitative trait loci (QTL) underlying SDS and SCN resistances. Two recombinant inbred line (RIL) populations were developed by crossing ‘A95-684043’, a high-yielding maturity group (MG) II line resistant to SCN, with ‘LS94-3207’ and ‘LS98-0582’ of MG IV, resistant to both F. virguliforme and SCN. Two hundred F7 derived recombinant inbred lines from each population AX19286 (A95-684043 × LS94-3207) and AX19287 (A95-684043 × LS98-0582) were screened for resistance to each pathogen under greenhouse conditions. Five hundred and eighty and 371 SNP markers were used for mapping resistance QTL in each population. In AX19286, one novel SCN resistance QTL was mapped to chromosome 8. In AX19287, one novel SDS resistance QTL was mapped to chromosome 17 and one novel SCN resistance QTL was mapped to chromosome 11. Previously identified additional SDS and SCN resistance QTL were also detected in the study. Lines possessing superior resistance to both pathogens were also identified and could be used as germplasm sources for breeding SDS- and SCN-resistant soybean cultivars.

Nonhost resistance is defined as the immunity of a plant species to all nonadapted pathogen species. Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 is nonhost to the oomycete plant pathogen Phytophthora sojae and the fungal plant pathogen Fusarium virguliforme that are pathogenic to soybean (Glycine max). Previously, we reported generating the pss1 mutation in the pen1-1 genetic background as well as genetic mapping and characterization of the Arabidopsis nonhost resistance Phytophthora sojae-susceptible gene locus, PSS1 In this study, we identified six candidate PSS1 genes by comparing single-nucleotide polymorphisms of (1) the bulked DNA sample of seven F2:3 families homozygous for the pss1 allele and (2) the pen1-1 mutant with Columbia-0. Analyses of T-DNA insertion mutants for each of these candidate PSS1 genes identified the At3g59640 gene encoding a glycine-rich protein as the putative PSS1 gene. Later, complementation analysis confirmed the identity of At3g59640 as the PSS1 gene. PSS1 is induced following P. sojae infection as well as expressed in an organ-specific manner. Coexpression analysis of the available transcriptomic data followed by reverse transcriptase-polymerase chain reaction suggested that PSS1 is coregulated with ATG8a (At4g21980), a core gene in autophagy. PSS1 contains a predicted single membrane-spanning domain. Subcellular localization study indicated that it is an integral plasma membrane protein. Sequence analysis suggested that soybean is unlikely to contain a PSS1-like defense function. Following the introduction of PSS1 into the soybean cultivar Williams 82, the transgenic plants exhibited enhanced resistance to F. virguliforme, the pathogen that causes sudden death syndrome.

High soil moisture usually favors soybean sudden death syndrome (SDS), caused by Fusarium virguliforme (Fv), but the effects of the duration of the flooding period and accompanying anaerobic conditions on the soybean-Fv interaction are not clear. Greenhouse studies were conducted using susceptible and resistant cultivars exposed to the following treatments: 3, 5, or 7 days of continuous flooding, repeated short-term flooding of 8 h/week for 3 weeks, and a no-flood check treatment. At 7, 14, and 21 days after flooding (DAF), seedlings in the no-flood, 3-day, and repeated short-term treatments showed the highest root rot and foliar symptom severity, whereas seedlings in the 7-day treatment showed the lowest severity. Fv inoculum density in soil was lowest in the 7-day flooding treatment. In a hydroponic system, the steady transcript levels of soybean defense genes and Fv candidate virulence genes were measured in response to different oxygen levels using qPCR. Fv-infected roots exposed to 12 h of anaerobic conditions showed down-regulation of the defense-related soybean genes Laccase, PR3, PR10, PAL, and CHS, and the Fv virulence genes pectate lyase (PL), and Fv homolog of the pisatin demethylase (PDA). Our study suggests that short-term flooding tends to increase SDS, while prolonged flooding negatively impacts SDS due to reduction of Fv density in soil. Moreover, anaerobic conditions down-regulate both soybean defense genes and Fv candidate virulence genes.

Using a combination of phenotypic screening and molecular, statistical, and linkage analyses, we have mapped a dominant soybean rust resistance gene in soybean PI 567104B. Asian soybean rust (SBR), caused by the fungus Phakopsora pachyrhizi Syd. and P. Syd., is one of the most economically important diseases that affect soybean production worldwide. A long-term strategy for minimizing the effects of SBR is the development of genetically resistant cultivars. The objectives of the study were to identify the location of a rust-resistance (Rpp) gene(s) in plant introduction (PI) 567104B, and to determine if the gene(s) in PI 567104B was different from previously mapped Rpp loci. The progeny of the cross of 'IAR 2001 BSR' × PI 567104B was phenotyped from field assays of the F 2:3 and F 4:5 generations and from a growth chamber assay of 253 F 5:6 recombinant inbred lines (RILs). For the growth chamber, the phenotyping was conducted by inoculation with a purified 2006 fungal isolate from Mississippi. A resistance gene locus on PI 567104B was mapped to a region containing the Rpp6 locus on chromosome 18. The high level of resistance of F 1 plants from two other crosses with PI 567104B as one of the parents indicated that the gene from PI 567104B was dominant. The interval containing the gene is flanked by the simple sequence repeat (SSR) markers Satt131 and Satt394, and includes the SSR markers BARCSOYSSR_18_0331 and BARCSOYSSR_18_0380. The results also indicated that the resistance gene from PI 567104B is different from the Rpp1 to the Rpp4 genes previously identified. To determine if the gene from PI 567104B is different from the Rpp6 gene from PI 567102B, additional research will be required.

Fusarium virguliforme is a soil borne pathogen that causes sudden death syndrome (SDS) in soybean plants. This pathogenic disease may result in severe soybean yield suppression and can cause serious economic harm. It has been shown that the FvTox1 toxin produced by the pathogen may be the root cause of foliar SDS. Anti-FvTox1 single-chain variable fragment antibody expressed in transgenic soybean plants was shown to neutralize the FvTox1 toxin involved in foliar SDS development. Here, we have investigated the binding affinities of FvTox1 with four FvTox1-interacting peptides of 7 to 12 amino acids identified from phage display libraries using both bioinformatics-based molecular simulations and label-free bioassays with a unique photonic crystal biosensor. Results from the molecular simulations have predicted the interaction energies and 3-dimensional (3D) structures of FvTox1 and FvTox1-interacting peptide complexes. Our label-free binding assays have further provided the interaction strength of FvTox1 with four different FvTox1-interacting peptides and experimentally confirmed the simulation results obtained from bioinformatics-based molecular calculations.

The causal agent of the soybean sudden death syndrome (SDS), Fusarium virguliforme, remains in infected roots and secretes toxins to cause foliar SDS. In this study we investigated the xylem sap, roots, and leaves of F. virguliforme-infected and -uninfected soybean seedlings for any changes in a set of over 3,000 metabolites following pathogen infection by conducting GC/MS and LC/MS/MS, and detected 273 biochemicals. Levels of many intermediates of the TCA cycle were reduced suggesting suppression of this metabolic pathway by the pathogen. There was an increased accumulation of peroxidated lipids in leaves of F. virguliforme-infected plants suggesting possible involvement of free radicals and lipoxygenases in foliar SDS development. Levels of both isoflavone conjugates and isoflavonoid phytoalexins were decreased in infected roots suggesting degradation of these metabolites by the pathogen to promote root necrosis. The levels of the plant immunity inducer pipecolic acid (Pip) and the plant hormone salicylic acid (SA) were significantly increased in xylem sap (in case of Pip) and leaves (in case of both Pip and SA) of F. virguliforme-infected soybean plants compared to the control plants. This suggests a major signaling role of Pip in inducing host defense responses in above ground parts of the F. virguliforme-infected soybean. Increased accumulation of pipecolic acid in foliar tissues was associated with the induction of GmALD1, the soybean homolog of Arabidopsis ALD1. This metabolomics study generated several novel hypotheses for studying the mechanisms of SDS development in soybean.

Soybean deploys multiple genetic mechanisms to confer tolerance to Fusarium virguliforme toxins. This study revealed that F. virguliforme culture filtrates could be used in mapping QTL underlying foliar SDS resistance. Sudden death syndrome (SDS) is a major soybean disease throughout most of the soybean growing regions in the world including the United States. The disease is caused by the fungal pathogen, Fusarium virguliforme (Fv). The fungus produces several toxins that are responsible for development of interveinal leaf chlorosis and necrosis, which are typical foliar SDS symptoms. Growing of resistant cultivars has been the most effective method in controlling the disease. The objective of the present study was to identify quantitative trait loci (QTL) underlying host responses of soybean to Fv toxins present in culture filtrates. To accomplish this objective, two recombinant inbred line (RIL) populations, AX19286 (A95-684043 × LS94-3207) and AX19287 (A95-684043 × LS98-0582), segregating for SDS resistance were evaluated for foliar symptom development by applying two screening protocols, the stem cutting and the root feeding assays. The AX19286 population revealed two major and seven minor QTL for SDS resistance. In the AX19287 population, we identified five major QTL and three minor QTL. The two QTL mapped to Chromosome 7 [molecular linkage group (MLG) M] and Chromosome 20 (MLG I) are most likely novel, and were detected through screening of the AX19287 population with stem cutting and root feeding assays, respectively. This study established that Fv culture filtrates could be employed in mapping QTL underlying foliar SDS resistance. The outcomes of the research also suggest that multiple genetic mechanisms might be used by soybean to overcome the toxic effects of the toxins secreted by the pathogen into culture filtrates.

This chapter outlines the principles upon which strategies for fungal and bacterial resistance may be based, to evaluate the types of genetic manipulations which may lead to increased resistance. Some fungal pathogens have acquired virulence by being able to detoxify the phytoalexins the host produces as a part of its defensive arsenal. A basis of information exists for engineering modified phytoalexin structures which may be resistant to detoxification, or for transferring a phytoalexin biosynthetic pathway from one plant to another which lacks that particular pathway. Transgenic plants expressing either novel proteins from foreign organisms or overexpressing a part of their own defensive arsenal have been engineered, tested in both laboratory and field situations, and evaluated for disease resistance. Pathogenesis-related proteins are low-molecular weight proteins which accumulate to significant levels in infected plant tissues. Plants, and indeed other organisms, may contain antimicrobial proteins not necessarily associated with induced defense responses, which are potential subjects for engineered protection strategies.

Type II transposable elements that use a cut-and-paste mechanism for jumping from one genomic region to another are ideal for use in tagging and cloning genes. Precise excision from an insertion site in a mutant gene leads to regaining the wild-type function. Thus, the function of a gene can be established based on the mutant phenotype and the regaining of the wild-type phenotype following precise excision of the element. Heterologous type II transposable elements including the Ac/Ds system from maize, the miniature inverted repeat system, mPing from rice, and the Tnt1 retrotransposon from tobacco have been successfully applied in functional analyses of soybean genes. Although several endogenous transposable elements have been identified in soybean, evidence of an active type II transposable element in soybean was largely lacking. We have previously reported the isolation of the type II soybean transposon Tgm9 from intron II of the dihyroflavonol-4-reductase 2 (DFR2) gene of the W4 locus. Tgm9 is an active element and produces variegated flowers through somatic excision. Excision of the Tgm9 element from the progenitor cells of flower buds results in genotypes with purple flowers that are known as germinal revertants. The element was discovered from a commercial soybean cultivar, and the line carrying the element was termed T322. The T322 genome contains only one active Tgm9 copy in the W4 locus. In a recent study, the utility of Tgm9 was assessed by studying a set of random germinal revertants. The new mutations created following excision of Tgm9 from DFR2 were evaluated using a transposon display assay. This study revealed that Tgm9 transposes to all 20 soybean chromosomes from its original site in the DFR2 gene. Although Tgm9 exhibited preferential transposition to a few genomic regions, across the entire genome 25.7% of the new Tgm9 mutants were detected in exon or intron sequences. Thus, Tgm9 is a suitable endogenous type II transposon to generate an indexed insertional mutant collection for functional characterization of most of the soybean genes.

In soybean, variegated flowers can be caused by somatic excision of the CACTA-type transposable element Tgm9 from Intron 2 of the DFR2 gene encoding dihydroflavonol-4-reductase of the anthocyanin pigment biosynthetic pathway. DFR2 was mapped to the W4 locus, where the allele containing Tgm9 was termed w4-m. In this study we have demonstrated that previously identified morphological mutants (three chlorophyll deficient mutants, one male sterile-female fertile mutant, and three partial female sterile mutants) were caused by insertion of Tgm9 following its excision from DFR2. Analyses of Tgm9 insertion sites among 105 independent mutants demonstrated that Tgm9 hops to all 20 soybean chromosomes from its original location on Chromosome 17. Some genomic regions are prone to increased Tgm9-insertions. Tgm9 transposed over 25% of the time into exon or intron sequences. Tgm9 is therefore suitable for generating an indexed insertional mutant collection for functional analyses of most soybean genes. Furthermore, desirable Tgm9-induced stable knockout mutants can be utilized in generating improved traits for commercial soybean cultivars.

Sudden death syndrome (SDS) is caused by the fungal pathogen, Fusarium virguliforme, and is a major threat to soybean production in North America. There are two major components of this disease: (i) root necrosis and (ii) foliar SDS. Root symptoms consist of root necrosis with vascular discoloration. Foliar SDS is characterized by interveinal chlorosis and leaf necrosis, and in severe cases by flower and pod abscission. A major toxin involved in initiating foliar SDS has been identified. Nothing is known about how root necrosis develops. In order to unravel the mechanisms used by the pathogen to cause root necrosis, the transcriptome of the pathogen in infected soybean root tissues of a susceptible cultivar, ‘Essex’, was investigated. The transcriptomes of the germinating conidia and mycelia were also examined. Of the 14,845 predicted F. virguliforme genes, we observed that 12,017 (81%) were expressed in germinating conidia and 12,208 (82%) in mycelia and 10,626 (72%) in infected soybean roots. Of the 10,626 genes induced in infected roots, 224 were transcribed only following infection. Expression of several infection-induced genes encoding enzymes with oxidation-reduction properties suggests that degradation of antimicrobial compounds such as the phytoalexin, glyceollin, could be important in early stages of the root tissue infection. Enzymes with hydrolytic and catalytic activities could play an important role in establishing the necrotrophic phase. The expression of a large number of genes encoding enzymes with catalytic and hydrolytic activities during the late infection stages suggests that cell wall degradation could be involved in root necrosis and the establishment of the necrotrophic phase in this pathogen.

Fusarium tucumaniae is the only known sexually reproducing species among the seven closely related fusaria that cause soybean sudden death syndrome (SDS) or bean root rot (BRR). In a previous study, laboratory mating of F. tucumaniae yielded recombinant ascospore progeny but required two mating-compatible strains, indicating that it is heterothallic. To assess the reproductive mode of the other SDS and BRR fusaria, and their potential for mating, whole-genome sequences of two SDS and one BRR pathogen were analyzed to characterize their mating type (MAT) loci. This bioinformatic approach identified a MAT1-1 idiomorph in F. virguliforme NRRL 22292 and MAT1-2 idiomorphs in F. tucumaniae NRRL 34546 and F. azukicola NRRL 54364. Alignments of the MAT loci were used to design PCR primers within the conserved regions of the flanking genes APN1 and SLA2, which enabled primer walking to obtain nearly complete sequences of the MAT region for six MAT1-1 and five MAT1-2 SDS/BRR fusaria. As expected, sequences of the highly divergent 4.7 kb MAT1-1 and 3.7 kb MAT1-2 idiomorphs were unalignable. However, sequences of the respective idiomorphs and those that flank MAT1-1 and MAT1-2 were highly conserved. In addition to three genes at MAT1-1 (MAT1-1-1, MAT1-1-2, MAT1-1-3) and two at MAT1-2 (MAT1-2-1, MAT1-2-3), the MAT loci of the SDS/BRR fusaria also include a putative gene predicted to encode for a 252 amino acid protein of unknown function. Alignments of the MAT1-1-3 and MAT1-2-1 sequences were used to design a multiplex PCR assay for the MAT loci. This assay was used to screen DNA from 439 SDS/BRR isolates, which revealed that each isolate possessed MAT1-1 or MAT1-2, consistent with heterothallism. Both idiomorphs were represented among isolates of F. azukicola, F. brasiliense, F. phaseoli and F. tucumaniae, whereas isolates of F. virguliforme and F. cuneirostrum were only MAT1-1 and F. crassistipitatum were only MAT1-2. Finally, nucleotide sequence data from the RPB1 and RPB2 genes were used to date the origin of the SDS/BRR group, which was estimated to have occurred about 0.75 Mya (95% HPD interval: 0.27, 1.68) in the mid-Pleistocene, long before the domestication of the common bean or soybean.

Disease resistance is an important crop trait that protects yield and farmers’ incomes. The objective of the soybean breeding program for disease resistance at Iowa State University (ISU) is to develop germplasm lines for Iowa. AR10SDS (Reg. No. GP-391, PI 669816) is the first sudden death syndrome (SDS) partially resistant line released by ISU, Project nos. 4403 and 0159 (Agronomy and Plant Pathology departments), ISU Research Foundation (ISURF) Docket #03264. AR10SDS is partially resistant to Fusarium virguliforme which causes SDS, and resistant to soybean cyst nematode (SCN) phenotypic race 3 (HG types 0 and 7) caused by Heterodera glycines. AR10SDS is a bulk of 60 F 3:8 plants uniform in plant and seed traits. The parentage is ‘Ripley’ × ‘IA1008’. Ripley is resistant to SDS. IA1008 is resistant to SCN. The cross and generations advances were made in Puerto Rico beginning in 2000, ISU research site at the Isabela Substation, University of Puerto Rico, Isabela, PR. Yield was evaluated from 2003 to 2006 in Iowa and in the northern U.S. regions. From 2003 to 2011, SDS resistance was evaluated under greenhouse conditions and in SDS-infested field soil. Soybean cyst nematode screening was conducted in 2005 to 2006 under greenhouse conditions. Three SDS resistance quantitative trait loci (QTLs) were inherited from Ripley, and two SCN resistance QTLs from IA1008. Seed yield is similar to IA1008 and significantly ( P = 0.05) better than ‘Parker’ and ‘Freeborn’. It is of early- to mid-maturity group I and adapted from 40 to 42° N latitude. AR10SDS may serve breeding programs for its dual disease resistance.

The soil borne fungus, Fusarium virguliforme, causes sudden death syndrome (SDS) in soybean, which is a serious foliar and root rot disease. The pathogen has never been isolated from the diseased foliar tissues; phytotoxins produced by the pathogen are believed to cause foliar SDS symptoms. One of these toxins, a 13.5-kDa acidic protein named FvTox1, has been hypothesized to interfere with photosynthesis in infected soybean plants and cause foliar SDS. The objective of this study is to determine if FvTox1 is involved in foliar SDS development. We created and studied five independent knockout fvtox1 mutants to study the function of FvTox1. We conducted Agrobacterium tumefaciens-mediated transformation to accomplish homologous recombination of FvTox1 with a hygromycin B resistance gene, hph, to generate the fvtox1 mutants. Approximately 40 hygromycin-resistant transformants were obtained from 10(6) conidial spores of the F. virguliforme Mont-1 isolate when the spores were co-cultivated with the A. tumefaciens EHA105 but not with LBA4044 strain carrying a recombinant binary plasmid, in which the hph gene encoding hygromycin resistance was flanked by 5'- and 3'-end FvTox1 sequences. We observed homologous recombination-mediated integration of hph into the FvTox1 locus among five independent fvtox1 mutants. In stem-cutting assays using cut soybean seedlings fed with cell-free F. virguliforme culture filtrates, the knockout fvtox1 mutants caused chlorophyll losses and foliar SDS symptoms, which were over twofold less than those caused by the virulent F. virguliforme Mont-1 isolate. Similarly, in root inoculation assays, more than a twofold reduction in foliar SDS development and chlorophyll losses was observed among the seedlings infected with the fvtox1 mutants as compared to the seedlings infected with the wild-type Mont-1 isolate. These results suggest that FvTox1 is a major virulence factor involved in foliar SDS development in soybean. It is expected that interference of the function of this toxin in transgenic soybean plants will lead to generation of SDS-resistant soybean cultivars.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice Fil: Geiser, David M.. State University of Pennsylvania; Estados Unidos Fil: Aoki, Takayuki. National Institute of Agrobiological Sciences; Japón Fil: Bacon, Charles W.. Toxicology and Mycotoxin Research; Grecia Fil: Baker, Scott E.. Pacific Northwest National Laboratory; Estados Unidos Fil: Bhattacharyya, Madan K.. University of Iowa; Estados Unidos Fil: Chulze, Sofia Noemi. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Scandiani, María Mercedes. Laboratorio Agrícola Río Paraná. San Pedro; Argentina Fil: Ploetz, Randy C.. University of Florida; Estados Unidos Fil: Proctor, Robert H.. NCAUR-ARS-USDA; Estados Unidos Fil: Rehner, Stephen A.. United States Department of Agriculture; Estados Unidos Fil: Robert, Vincent A. R. G.. Fungal Biodiversity Center; Países Bajos Fil: Rooney, Alejandro P.. NCAUR-ARS-USDA; Estados Unidos Fil: Salleh, Baharuddin bin. University of Malaysia; Malasia Fil: Scauflaire, Jonathan. Université Catholique de Louvain; Bélgica Fil: Short, Dylan P. G.. University of California; Estados Unidos Fil: Steenkamp, Emma. University of Pretoria; Sudáfrica Fil: Suga, Haruhisa. Gifu University; Japón Fil: Summerell, Brett A.. The Royal Botanic Garden Sydney; Australia Fil: Sutton, Deanna A.. University of Texas; Estados Unidos Fil: Thrane, Ulf. Technical University of Denmark; Dinamarca Fil: Trail, Francis. Michigan State University; Estados Unidos Fil: Van Diepeningen, Anne. Fungal Biodiversity Center; Países Bajos Fil: VanEtten, Hans D.. University of Arizona; Estados Unidos Fil: Viljoen, Altus. University of Stellenbosch; Sudáfrica Fil: Waalwijk, Cees. Wageningen University; Países Bajos Fil: Ward, Todd J.. NCAUR-ARS-USDA. Pretoria; Sudáfrica Fil: Wingfield, Michael J.. University of Pretoria; Sudáfrica Fil: Xu, Jin-Rong. Purdue University; Estados Unidos Fil: Yang, Xiao-Bing. University of Iowa; Estados Unidos Fil: Yli-Mattila, Tapani. University of Turku; Finlandia Fil: Zhang, Ning. Rutgers University; Estados Unidos

Supernumerary chromosome segments are known to harbor different transposons from their essential counterparts. The aim of this study was to investigate the role of transposons in the origin and evolution of supernumerary segments in the asexual fungal pathogen Fusarium virguliforme. We compared the genomes of 11 isolates comprising six Fusarium species that cause soybean sudden death syndrome (SDS) or bean root rot (BRR), and identified significant levels of genetic variation in A+T-rich repeat blocks of the essential chromosomes and in A+T-neutral regions of the supernumerary segments. The A+T-rich repeat blocks in the essential chromosomes were highly variable between F. virguliforme and non-F. virguliforme isolates, but were scarcely variable between F. virguliforme isolates. The A+T-neutral regions in the supernumerary segments, however, were highly variable between F. virguliforme isolates, with a statistically significant number (21 standard deviations above the mean) of single nucleotide polymorphisms (SNPs). And supernumerary sequence types and rearrangement patterns of some F. virguliforme isolates were present in an isolate of F. cuneirostrum but not in the other F. virguliforme isolates. The most variable and highly expressed region in the supernumerary segments contained an active DNA transposon that was a most conserved match between F. virguliforme and the unrelated fungus Tolypocladium inflatum. This transposon was absent from two of the F. virguliforme isolates. Furthermore, transposons in the supernumerary segments of some F. virguliforme isolates were present in non-F. virguliforme isolates, but were absent from the other F. virguliforme isolates. Two supernumerary P450 enzymes were 43% and 57% identical to their essential counterparts. This study has raised the possibility that transposons generate genetic variation in supernumerary chromosome segments by frequent horizontal transfer within and between closely related species.

This paper reports a highly economical and accessible approach to generate different discrete relative humidity conditions in spatially separated wells of a modified multi-well plate for humidity assay of plant-pathogen interactions with good throughput. We demonstrated that a discrete humidity gradient could be formed within a few minutes and maintained over a period of a few days inside the device. The device consisted of a freeway channel in the top layer, multiple compartmented wells in the bottom layer, a water source, and a drying agent source. The combinational effects of evaporation, diffusion, and convection were synergized to establish the stable discrete humidity gradient. The device was employed to study visible and molecular disease phenotypes of soybean in responses to infection by Phytophthora sojae, an oomycete pathogen, under a set of humidity conditions, with two near-isogenic soybean lines, Williams and Williams 82, that differ for a Phytophthora resistance gene (Rps1-k). Our result showed that at 63% relative humidity, the transcript level of the defense gene GmPR1 was at minimum in the susceptible soybean line Williams and at maximal level in the resistant line Williams 82 following P. sojae CC5C infection. In addition, we investigated the effects of environmental temperature, dimensional and geometrical parameters, and other configurational factors on the ability of the device to generate miniature humidity environments. This work represents an exploratory effort to economically and efficiently manipulate humidity environments in a space-limited device and shows a great potential to facilitate humidity assay of plant seed germination and development, pathogen growth, and plant-pathogen interactions. Since the proposed device can be easily made, modified, and operated, it is believed that this present humidity manipulation technology will benefit many laboratories in the area of seed science, plant pathology, and plant-microbe biology, where humidity is an important factor that influences plant disease infection, establishment, and development.

Fusarium virguliforme causes the serious disease sudden death syndrome (SDS) in soybean. Host resistance to this pathogen is partial and is encoded by a large number of quantitative trait loci, each conditioning small effects. Breeding SDS resistance is therefore challenging and identification of single-gene encoded novel resistance mechanisms is becoming a priority to fight this devastating this fungal pathogen. In this transcriptomic study we identified a few putative soybean defense genes, expression of which is suppressed during F. virguliforme infection. The F. virguliforme infection-suppressed genes were broadly classified into four major classes. The steady state transcript levels of many of these genes were suppressed to undetectable levels immediately following F. virguliforme infection. One of these classes contains two novel genes encoding ankyrin repeat-containing proteins. Expression of one of these genes, GmARP1, during F. virguliforme infection enhances SDS resistance among the transgenic soybean plants. Our data suggest that GmARP1 is a novel defense gene and the pathogen presumably suppress its expression to establish compatible interaction.

In soybean, the W4 gene encoding dihydroflavonol- 4-reductase controls anthocyanin pigment biosynthesis in flow- ers. The mutant allele, w4-m, is characterized by variegated flowers and was evolved from the insertion of an endogenous transposable element, Tgm9 ,i n intron II of theW4 gene. In the w4-m mutant line, reversion of the unstable allele from varie- gated to normal purple flower in revertants would indicate Tgm9's excision accompanied by its insertion into a second locus. We identified a male-sterile, female-sterile mutant from such germinal revertant bearing purple flowers. The objectives of our investigation were to map the sterility locus, identify candidate genes for the male-fertile, female-fertile phenotype, and then determine if sterility is associated with the insertion of Tgm9in the sterility locus. We usedbulked segregant analysisto map the locus to molecular linkage group J (chromosome 16). Fine mapping enabled us to flank the locus to a 62-kb region that contains only five predicted genes. One of the genes in that region, Glyma16g07850.1, codes for a helicase. A rice homolog of this gene has been shown to control crossing over and fertility phenotype. Thus, Glyma16g07850.1 is most likely the gene regulating the male and female fertility phenotype in soybean. DNA blot analysis of the segregating individuals for Tgm9 showed perfect association between sterility and the presence of the transposon. Most likely, the sterility mutation was caused by the insertion of Tgm9. The transposable element should facilitate identification of the male- and female-fertility gene. Characterization of the fertility gene will provide vital molecular insight on the reproductive biology of soybean and other plants.