Whole-genome sequencing uncovers the structural and transcriptomic landscape of hexaploid wheat/Ambylopyrum muticum introgression lines.

Wheat is a globally vital crop, but its limited genetic variation creates a challenge for breeders aiming to maintain or accelerate agricultural improvements over time. Introducing novel genes and alleles from wheat's wild relatives into the wheat breeding pool via introgression lines is an important component of overcoming this low variation but is constrained by poor genomic resolution and limited understanding of the genomic impact of introgression breeding programmes. By sequencing 17 hexaploid wheat/Ambylopyrum muticum introgression lines and the parent lines, we have precisely pinpointed the borders of introgressed segments, most of which occur within genes. We report a genome assembly and annotation of Am. muticum that has facilitated the identification of Am. muticum resistance genes commonly introgressed in lines resistant to stripe rust. Our analysis has identified an abundance of structural disruption and homoeologous pairing across the introgression lines, likely caused by the suppressed Ph1 locus. mRNAseq analysis of six of these introgression lines revealed that novel introgressed genes are rarely expressed and those that directly replace a wheat orthologue have a tendency towards downregulation, with no discernible compensation in the expression of homoeologous copies. This study explores the genomic impact of introgression breeding and provides a schematic that can be followed to characterize introgression lines and identify segments and candidate genes underlying the phenotype. This will facilitate more effective utilization of introgression pre-breeding material in wheat breeding programmes.

Wheat Virus Identification Within Infected Tissue Using Nanopore Sequencing Technology.

Viral diseases are a limiting factor to wheat production. Viruses are difficult to diagnose in the early stages of disease development and are often confused with nutrient deficiencies or other abiotic problems. Immunological methods are useful to identify viruses, but specific antibodies may not be available or require high virus titer for detection. In 2015 and 2017, wheat plants containing Wheat streak mosaic virus (WSMV) resistance gene, Wsm2, were found to have symptoms characteristic of WSMV. Serologically, WSMV was detected in all four samples. Additionally, High Plains wheat mosaic virus (HPWMoV) was also detected in one of the samples. Barley yellow dwarf virus (BYDV) was not detected, and a detection kit was not readily available for Triticum mosaic virus (TriMV). Initially, cDNA cloning and Sanger sequencing were used to determine the presence of WSMV; however, the process was time-consuming and expensive. Subsequently, cDNA from infected wheat tissue was sequenced with single-strand, Oxford Nanopore sequencing technology (ONT). ONT was able to confirm the presence of WSMV. Additionally, TriMV was found in all of the samples and BYDV in three of the samples. Deep coverage sequencing of full-length, single-strand WSMV revealed variation compared with the WSMV Sidney-81 reference strain and may represent new variants which overcome Wsm2. These results demonstrate that ONT can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants.

Highly predictive SNP markers for efficient selection of the wheat leaf rust resistance gene Lr16.

BACKGROUND: Lr16 is a widely deployed leaf rust resistance gene in wheat (Triticum aestivum L.) that is highly effective against the North American Puccinia triticina population when pyramided with the gene Lr34. Lr16 is a seedling leaf rust resistance gene conditioning an incompatible interaction with a distinct necrotic ring surrounding the uredinium. Lr16 was previously mapped to the telomeric region of the short arm of wheat chromosome 2B. The goals of this study were to develop numerous single nucleotide polymorphism (SNP) markers for the Lr16 region and identify diagnostic gene-specific SNP marker assays for marker-assisted selection (MAS). RESULTS: Forty-three SNP markers were developed and mapped on chromosome 2BS tightly linked with the resistance gene Lr16 across four mapping populations representing a total of 1528 gametes. Kompetitive Allele Specific PCR (KASP) assays were designed for all identified SNPs. Resistance gene analogs (RGAs) linked with the Lr16 locus were identified and RGA-based SNP markers were developed. The diagnostic potential of the SNPs co-segregating with Lr16 was evaluated in a diverse set of 133 cultivars and breeding lines. Six SNP markers were consistent with the Lr16 phenotype and are accurately predictive of Lr16 for all wheat lines/cultivars in the panel. CONCLUSIONS: Lr16 was mapped relative to SNP markers in four populations. Six SNP markers exhibited high quality clustering in the KASP assay and are suitable for MAS of Lr16 in wheat breeding programs.

Conserved loci of leaf and stem rust fungi of wheat share synteny interrupted by lineage-specific influx of repeat elements.

BACKGROUND: Wheat leaf rust (Puccinia triticina Eriks; Pt) and stem rust fungi (P. graminis f.sp. tritici; Pgt) are significant economic pathogens having similar host ranges and life cycles, but different alternate hosts. The Pt genome, currently estimated at 135 Mb, is significantly larger than Pgt, at 88 Mb, but the reason for the expansion is unknown. Three genomic loci of Pt conserved proteins were characterized to gain insight into gene content, genome complexity and expansion. RESULTS: A bacterial artificial chromosome (BAC) library was made from P. triticina race 1, BBBD and probed with Pt homologs of genes encoding two predicted Pgt secreted effectors and a DNA marker mapping to a region of avirulence. Three BACs, 103 Kb, 112 Kb, and 166 Kb, were sequenced, assembled, and open reading frames were identified. Orthologous genes were identified in Pgt and local conservation of gene order (microsynteny) was observed. Pairwise protein identities ranged from 26 to 99%. One Pt BAC, containing a RAD18 ortholog, shares syntenic regions with two Pgt scaffolds, which could represent both haplotypes of Pgt. Gene sequence is diverged between the species as well as within the two haplotypes. In all three BAC clones, gene order is locally conserved, however, gene shuffling has occurred relative to Pgt. These regions are further diverged by differing insertion loci of LTR-retrotransposon, Gypsy, Copia, Mutator, and Harbinger mobile elements. Uncharacterized Pt open reading frames were also found; these proteins are high in lysine and similar to multiple proteins in Pgt. CONCLUSIONS: The three Pt loci are conserved in gene order, with a range of gene sequence divergence. Conservation of predicted haustoria expressed secreted protein genes between Pt and Pgt is extended to the more distant poplar rust, Melampsora larici-populina. The loci also reveal that genome expansion in Pt is in part due to higher occurrence of repeat-elements in this species.

A unique wheat disease resistance-like gene governs effector-triggered susceptibility to necrotrophic pathogens.

Plant disease resistance is often conferred by genes with nucleotide binding site (NBS) and leucine-rich repeat (LRR) or serine/threonine protein kinase (S/TPK) domains. Much less is known about mechanisms of susceptibility, particularly to necrotrophic fungal pathogens. The pathogens that cause the diseases tan spot and Stagonospora nodorum blotch on wheat produce effectors (host-selective toxins) that induce susceptibility in wheat lines harboring corresponding toxin sensitivity genes. The effector ToxA is produced by both pathogens, and sensitivity to ToxA is governed by the Tsn1 gene on wheat chromosome arm 5BL. Here, we report the cloning of Tsn1, which was found to have disease resistance gene-like features, including S/TPK and NBS-LRR domains. Mutagenesis revealed that all three domains are required for ToxA sensitivity, and hence disease susceptibility. Tsn1 is unique to ToxA-sensitive genotypes, and insensitive genotypes are null. Sequencing and phylogenetic analysis indicated that Tsn1 arose in the B-genome diploid progenitor of polyploid wheat through a gene-fusion event that gave rise to its unique structure. Although Tsn1 is necessary to mediate ToxA recognition, yeast two-hybrid experiments suggested that the Tsn1 protein does not interact directly with ToxA. Tsn1 transcription is tightly regulated by the circadian clock and light, providing further evidence that Tsn1-ToxA interactions are associated with photosynthesis pathways. This work suggests that these necrotrophic pathogens may thrive by subverting the resistance mechanisms acquired by plants to combat other pathogens.

Brome mosaic virus detected in Kansas wheat co-infected with other common wheat viruses.

Wheat breeders are developing new virus-resistant varieties; however, it is assumed that only a few viruses or well-known viruses are present in the field. New sequencing technology is allowing for better determination of natural field virus populations. For three years, 2019-2021, Kansas wheat field surveys were conducted to determine the constituents of natural field virus populations using nanopore sequencing. During analysis, brome mosaic virus (BMV) was identified for the first time in Kansas but was in association with other wheat viruses. Brome mosaic virus was identified from 29 out of 47 different Kansas counties sampled and 44% of the total samples. BMV was found co-infected with wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) in 27.8% of the samples, with WSMV only (13.9%) and co-infected with WSMV + TriMV + High Plains wheat mosaic emaravirus (HPWMoV) (13.9%). RNA genomes of Kansas BMV isolates had 99.4 to 100% nucleotide and amino acid sequence identity, respectively, to each other. RNA2a possessed relatively high divergence (π = 0.01) compared to RNA1a and RNA3a (π = 0.004). Coding regions of all BMV RNAs were considered negative for purifying selection pressure as nonsynonymous and synonymous nucleotide ratio was less than one (dNs/dS >1). The identification of BMV in Kansas virus populations adds another layer of complexity to plant breeding. This work provides information to improve tools to aid in monitoring, detecting, and determining the variation within BMV.

Population genomics of Puccinia graminis f.sp. tritici highlights the role of admixture in the origin of virulent wheat rust races.

Puccinia graminis f.sp. tritici (Pgt) causes stem rust disease in wheat that can result in severe yield losses. The factors driving the evolution of its virulence and adaptation remain poorly characterized. We utilize long-read sequencing to develop a haplotype-resolved genome assembly of a U.S. isolate of Pgt. Using Pgt haplotypes as a reference, we characterize the structural variants (SVs) and single nucleotide polymorphisms in a diverse panel of isolates. SVs impact the repertoire of predicted effectors, secreted proteins involved in host-pathogen interaction, and show evidence of purifying selection. By analyzing global and local genomic ancestry we demonstrate that the origin of 8 out of 12 Pgt clades is linked with either somatic hybridization or sexual recombination between the diverged donor populations. Our study shows that SVs and admixture events appear to play an important role in broadening Pgt virulence and the origin of highly virulent races, creating a resource for studying the evolution of Pgt virulence and preventing future epidemic outbreaks.

Occurrence and Yield Effects of Wheat Infected with Triticum mosaic virus in Kansas.

Triticum mosaic virus (TriMV) infects wheat (Triticum aestivum) in the Great Plains region of the United States. This study determined the occurrence of TriMV at three locations over 3 years and yield effects of wheat mechanically infected with TriMV. Wheat infection with TriMV, Wheat streak mosaic virus (WSMV), and the High Plains virus (HPV) was verified using enzyme-linked immunosorbent assay. Both wheat singly infected with TriMV and doubly infected with TriMV and WSMV occurred at three, two, and one locations in 2007, 2008, and 2009, respectively. Wheat singly infected with HPV occurred at one and two locations in 2008 and 2009, respectively. Wheat doubly infected with WSMV and HPV occurred at one location in 2008 and 2009. Infection with TriMV declined at two locations each year and, at the third location, it increased the second year and was not detected the third year. WSMV infection increased, except for a decline the third year at one location. In contrast to 3.0% infection of wheat with TriMV and WSMV at one location, 85% of the wheat 1.6 km from that site was infected with TriMV and WSMV in 2009. Infection of wheat with TriMV caused significant yield and volume weight reductions in Danby, RonL, and Jagalene but not KS96HW10-3 wheat.

Triticum mosaic virus Isolates in the Southern Great Plains.

In 2006, a previously unknown wheat (Triticum aestivum) virus was discovered in Western Kansas and given the name Triticum mosaic virus (TriMV). TriMV has since been found in wheat samples isolated all across the Great Plains. Even though it can infect singularly, TriMV is mostly found with Wheat streak mosaic virus (WSMV) as a co-infection. The potential for TriMV to cause economic loss is significant, but very little is known about the virus. The objective of this study was to survey the TriMV population for genetic variation by nucleotide sequencing of isolates across a geographical region. A secondary objective was to characterize the WSMV isolates that are being co-transmitted with TriMV. Fourteen different TriMV isolations were taken from locations in Texas, Oklahoma, and Kansas, and the coat protein cDNA was sequenced. Thirteen nucleotide differences were found in the TriMV isolates, of which three induce amino acid changes. WSMV isolates had 65 nucleotide changes when compared to WSMV Sydney81. Our results indicate the TriMV virus population has minimal amounts of sequence variation and no singular WSMV genotype is specifically associated with TriMV co-infection. Based on the isolates analyzed, it appears that the field population of TriMV is very homogeneous.

Whole-genome sequencing of multiple isolates of Puccinia triticina reveals asexual lineages evolving by recurrent mutations.

The wheat leaf rust fungus, Puccinia triticina Erikss., is a worldwide pathogen of tetraploid durum and hexaploid wheat. Many races of P. triticina differ for virulence to specific leaf rust resistance genes and are found in most wheat-growing regions of the world. Wheat cultivars with effective leaf rust resistance exert selection pressure on P. triticina populations for virulent race types. The objectives of this study were to examine whole-genome sequence data of 121 P. triticina isolates and to gain insight into race evolution. The collection included isolates comprising of many different race phenotypes collected worldwide from common and durum wheat. One isolate from wild wheat relative Aegilops speltoides and two from Ae. cylindrica were also included for comparison. Based on 121,907 informative variants identified relative to the reference Race 1-1 genome, isolates were clustered into 11 major lineages with 100% bootstrap support. The isolates were also grouped based on variation in 1311 predicted secreted protein genes. In gene-coding regions, all groups had high ratios of nonsynonymous to synonymous mutations and nonsense to readthrough mutations. Grouping of isolates based on two main variation principle components for either genome-wide variation or variation just within the secreted protein genes, indicated similar groupings. Variants were distributed across the entire genome, not just within the secreted protein genes. Our results suggest that recurrent mutation and selection play a major role in differentiation within the clonal lineages.

Resistance to wheat rusts identified in wheat/Amblyopyrum muticum chromosome introgressions.

Wheat (Triticum aestivum L.) rusts are a worldwide production problem. Plant breeders have used genetic resistance to combat these fungi. However, single-gene resistance is rapidly overcome as a result of frequent occurrence of new virulent fungal strains. Thus, a supply of new resistance sources is continually needed, and new resistance sources are limited within hexaploid wheat genetic stocks. Wild relatives are able to be a resource for new resistance genes but are hindered because of chromosome incapability with domesticated wheats. Twenty-eight double-haploid hexaploid wheat/Amblyopyrum muticum (Boiss.) Eig introgression lines, with introgressions covering the majority of the T genome, were evaluated for resistance to Puccinia triticina Erikss., P. graminis Pers.:Pers. f.sp. tritici Erikss. & E. Henning, and P. striiformis Westend. f.sp. tritici Erikss.. At the seedling level, four lines were resistant to races of P. triticina, six lines were resistant to P. graminis, and 15 lines were resistant to P. striiformis. At the adult stage, 16 lines were resistant to P. triticina. Line 355 had resistance to all three rusts and line 161 had resistance to all tested races of P. triticina. Some of these lines will require further work to reduce the size of the introgressed segment; however, lines 92 and 355 have very small fragments and can be used directly as new resistance donors.

A Horizontal Gene Transfer Led to the Acquisition of a Fructan Metabolic Pathway in a Gall Midge.

Animals are thought to use only glucose polymers (glycogen) as energy reserve, whereas both glucose (starch) and fructose polymers (fructans) are used by microbes and plants. Here, it is reported that the gall midge Mayetiola destructor, and likely other herbivorous animal species, gained the ability to utilize dietary fructans directly as storage polysaccharides by a single horizontal gene transfer (HGT) of bacterial levanase/inulinase gene followed by gene expansion and differentiation. Multiple genes encoding levanases/inulinases have their origin in a single HGT event from a bacterium and they show high expression levels and enzymatic activities in different tissues of the gall midge, including nondigestive fat bodies and eggs, both of which contained significant amounts of fructans. This study provides evidence that animals can also use fructans as energy reserve by incorporating bacterial genes in their genomes.

A BAC-based physical map of the Hessian fly genome anchored to polytene chromosomes.

BACKGROUND: The Hessian fly (Mayetiola destructor) is an important insect pest of wheat. It has tractable genetics, polytene chromosomes, and a small genome (158 Mb). Investigation of the Hessian fly presents excellent opportunities to study plant-insect interactions and the molecular mechanisms underlying genome imprinting and chromosome elimination. A physical map is needed to improve the ability to perform both positional cloning and comparative genomic analyses with the fully sequenced genomes of other dipteran species. RESULTS: An FPC-based genome wide physical map of the Hessian fly was constructed and anchored to the insect's polytene chromosomes. Bacterial artificial chromosome (BAC) clones corresponding to 12-fold coverage of the Hessian fly genome were fingerprinted, using high information content fingerprinting (HIFC) methodology, and end-sequenced. Fluorescence in situ hybridization (FISH) co-localized two BAC clones from each of the 196 longest contigs on the polytene chromosomes. An additional 70 contigs were positioned using a single FISH probe. The 266 FISH mapped contigs were evenly distributed and covered 60% of the genome (95,668 kb). The ends of the fingerprinted BACs were then sequenced to develop the capacity to create sequenced tagged site (STS) markers on the BACs in the map. Only 3.64% of the BAC-end sequence was composed of transposable elements, helicases, ribosomal repeats, simple sequence repeats, and sequences of low complexity. A relatively large fraction (14.27%) of the BES was comprised of multi-copy gene sequences. Nearly 1% of the end sequence was composed of simple sequence repeats (SSRs). CONCLUSION: This physical map provides the foundation for high-resolution genetic mapping, map-based cloning, and assembly of complete genome sequencing data. The results indicate that restriction fragment length heterogeneity in BAC libraries used to construct physical maps lower the length and the depth of the contigs, but is not an absolute barrier to the successful application of the technology. This map will serve as a genomic resource for accelerating gene discovery, genome sequencing, and the assembly of BAC sequences. The Hessian fly BAC-clone assembly, and the names and positions of the BAC clones used in the FISH experiments are publically available at (http://genome.purdue.edu/WebAGCoL/Hfly/WebFPC/).

The complete genome sequence of Triticum mosaic virus, a new wheat-infecting virus of the High Plains.

The genome of Triticum mosaic virus (TriMV), a recently discovered mite-transmitted wheat potyvirus, was sequenced, characterized, and compared to other members of the family Potyviridae. TriMV has a single mRNA strand of 10,266 nucleotides with a predicted polyprotein consisting of 3,112 peptides. Protein alignments of the coat protein demonstrate that TriMV has 45.9% identity to Sugarcane streak mosaic virus strain AP (SCSMV-AP), but shares only 23.2% identity to Wheat streak mosaic virus. Although TriMV is mite-transmitted and could be placed in the genus Tritimovirus, it is significantly divergent and should be placed in the newly proposed genus Susmovirus.

Identification of the Wheat Curl Mite as the Vector of Triticum mosaic virus.

Triticum mosaic virus (TriMV) is a newly discovered virus found infecting wheat (Triticum aestivum) in Kansas. This study was conducted to determine if the wheat curl mite (WCM, Aceria tosichella) and the bird cherry oat aphid (Rhopalosiphum padi) could transmit TriMV. Using different sources of WCM and two different isolates of TriMV, we were able to show the WCM is the vector of TriMV. Field analysis by enzyme-linked immunosorbent assay (ELISA) demonstrated natural infection patterns of wheat infected with TriMV, Wheat streak mosaic virus (WSMV), or both TriMV and WSMV, putatively infected by viruliferous WCM from a volunteer source growing adjacent to the wheat. Moreover, by single WCM transfers using WCM obtained from different wheat plants naturally infected with TriMV and WSMV and naturally infested with WCM, we showed that these WCM also transmitted TriMV only to wheat or transmitted both TriMV and WSMV to wheat. The infection rates of wheat with TriMV only using WCM transmission was low in both laboratory and field analyses. However, field analyses by ELISA showed that levels of infection of wheat by both TriMV and WSMV were high. No transmission of TriMV to wheat by R. padi occurred in our studies.

Triticum mosaic virus: A New Virus Isolated from Wheat in Kansas.

In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses. The coat protein was estimated from SDS-PAGE to have a mass of approximately 35 kDa. Its amino acid sequence, as deduced from DNA sequencing of cloned, reverse-transcribed viral RNA and separately determined by time-of-flight mass spectrometry, was most closely related (49% similarity) to Sugarcane streak mosaic virus, a member of the Tritimovirus genus of the family Potyviridae. The virus gave strong positive reactions during enzyme-linked immunosorbent assays using polyclonal antibodies raised against purified preparations of the cognate virus but gave consistent negative reactions against antibodies to Wheat streak mosaic virus (WSMV), other wheat potyviruses, and the High Plains virus. When the virus was inoculated on the WSMV-resistant wheat cv. RonL, systemic symptoms appeared and plant growth was diminished significantly in contrast with WSMV-inoculated RonL. Taken together, the data support consideration of this virus as a new potyvirus, and the name Triticum mosaic virus (TriMV) is proposed.

Wheat differential gene expression induced by different races of Puccinia triticina.

Puccinia triticina, the causal agent of wheat leaf rust, causes significant losses in wheat yield and quality each year worldwide. During leaf rust infection, the host plant recognizes numerous molecules, some of which trigger host defenses. Although P. triticina reproduces clonally, there is still variation within the population due to a high mutation frequency, host specificity, and environmental adaptation. This study explores how wheat responds on a gene expression level to different P. triticina races. Six P. triticina races were inoculated onto a susceptible wheat variety and samples were taken at six days post inoculation, just prior to pustule eruption. RNA sequence data identified 63 wheat genes differentially expressed between the six races. A time course, conducted over the first seven days post inoculation, was used to examine the expression pattern of 63 genes during infection. Forty-seven wheat genes were verified to have differential expression. Three common expression patterns were identified. In addition, two genes were associated with race specific gene expression. Differential expression of an ER molecular chaperone gene was associated with races from two different P. triticina lineages. Also, differential expression in an alanine glyoxylate aminotransferase gene was associated with races with virulence shifts for leaf rust resistance genes.

Molecular characterization of the major wheat domestication gene Q.

The Q gene is largely responsible for the widespread cultivation of wheat because it confers the free-threshing character. It also pleiotropically influences many other domestication-related traits such as glume shape and tenacity, rachis fragility, spike length, plant height, and spike emergence time. We isolated the Q gene and verified its identity by analysis of knockout mutants and transformation. The Q gene has a high degree of similarity to members of the AP2 family of transcription factors. The Q allele is more abundantly transcribed than q, and the two alleles differ for a single amino acid. An isoleucine at position 329 in the Q protein leads to an abundance of homodimer formation in yeast cells, whereas a valine in the q protein appears to limit homodimer formation. Ectopic expression analysis allowed us to observe both silencing and overexpression effects of Q. Rachis fragility, glume shape, and glume tenacity mimicked the q phenotype in transgenic plants exhibiting post-transcriptional silencing of the transgene and the endogenous Q gene. Variation in spike compactness and plant height were associated with the level of transgene transcription due to the dosage effects of Q. The q allele is the more primitive, and the mutation that gave rise to Q occurred only once leading to the world's cultivated wheats.

Comparative Analysis Highlights Variable Genome Content of Wheat Rusts and Divergence of the Mating Loci.

Three members of the Puccinia genus, Pucciniatriticina (Pt), Pstriiformis f.sp. tritici (Pst), and Pgraminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi, and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; in comparison, repeats occupy 31.5% for Pst and 36.5% for Pgt We find all three genomes are highly heterozygous, with Pst [5.97 single nucleotide polymorphisms (SNPs)/kb] nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt Of 1358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host-induced gene silencing (HIGS) of the HD and STE3 alleles reduced wheat host infection.

Cloning and characterization of cDNAS encoding carboxypeptidase-like proteins from the gut of Hessian fly larvae [Mayetiola destructor (Say)].

Transcriptomic analysis of the gut from Hessian fly larvae [Mayetiola destructor (Say)] identified nine cDNA clones that encode different carboxypeptidase-like proteins. Sequence comparison revealed that five of the nine cDNAs encoded very similar proteins with amino acid sequence identity over 96%. The other four cDNAs encoded diversified proteins with amino acid sequence identity less than 60%. Further sequence comparison with well characterized carboxypeptidases from other organisms revealed that these cDNAs encoded MDCP (M. destructor carboxypeptidase)-A1, MDCP-A2, MDCP-B, MDCP-BL, and MDCP-D. All residues characteristic of metallocarboxypeptidases including the HXXE motif were conserved among members. Northern blot analysis revealed various expression patterns for different gene groups in different developmental stages of M. destructor, suggesting that individual carboxypeptidases perform specific functions or have different specificities. Enzymatic activity assays demonstrated that both carboxypeptidases A and B are predominant in the larval stage, the only feeding stage of M. destructor, indicating a role in food digestion. The digestive role is further supported by the fact that 80% of the enzymatic activity in larvae occurred in the gut. Among these two types of enzymes, the activity of carboxypeptidase A was at least four times higher than that of carboxypeptidase B under the same conditions, suggesting that carboxypeptidase A is the major digestive enzyme in the gut of M. destructor larvae.