slag: A program for seeded local assembly of genes in complex genomes.

Although finished genomes have become more common, there is still a need for assemblies of individual genes or chromosomal regions when only unassembled reads are available. slag (Seeded Local Assembly of Genes) fulfils this need by performing iterative local assembly based on cycles of matching-read retrieval with blast and assembly with cap3, phrap, spades, canu or unicycler. The target sequence can be nucleotide or protein. Read fragmentation allows slag to use phrap or cap3 to assemble long reads at lower coverage (e.g., 5×) than is possible with canu or unicycler. In simple, nonrepetitive genomes, a slag assembly can cover a whole chromosome, but in complex genomes the growth of target-matching contigs is limited as additional reads are consumed by consensus contigs consisting of repetitive elements. Apart from genomic complexity, contig length and correctness depend on read length and accuracy. With pyrosequencing or Illumina reads, slag-assembled contigs are accurate enough to allow design of PCR primers, while contigs assembled from Oxford Nanopore or pre-HiFi Pacific Biosciences long reads are generally only accurate enough to design baiting sequences for further targeted sequencing. In an application with real reads, slag successfully extended sequences for four wheat genes, which were verified by cloning and Sanger sequencing of overlapping amplicons. slag is a robust alternative to atram2 for local assemblies, especially for read sets with less than 20× coverage. slag is freely available at https://github.com/cfcrane/SLAG.

Comparative Hessian Fly Larval Transcriptomics Provides Novel Insight into Host and Nonhost Resistance.

The Hessian fly is a destructive pest of wheat. Employing additional molecular strategies can complement wheat's native insect resistance. However, this requires functional characterization of Hessian-fly-responsive genes, which is challenging because of wheat genome complexity. The diploid Brachypodium distachyon (Bd) exhibits nonhost resistance to Hessian fly and displays phenotypic/molecular responses intermediate between resistant and susceptible host wheat, offering a surrogate genome for gene characterization. Here, we compared the transcriptomes of Biotype L larvae residing on resistant/susceptible wheat, and nonhost Bd plants. Larvae from susceptible wheat and nonhost Bd plants revealed similar molecular responses that were distinct from avirulent larval responses on resistant wheat. Secreted salivary gland proteins were strongly up-regulated in all larvae. Genes from various biological pathways and molecular processes were up-regulated in larvae from both susceptible wheat and nonhost Bd plants. However, Bd larval expression levels were intermediate between larvae from susceptible and resistant wheat. Most genes were down-regulated or unchanged in avirulent larvae, correlating with their inability to establish feeding sites and dying within 4-5 days after egg-hatch. Decreased gene expression in Bd larvae, compared to ones on susceptible wheat, potentially led to developmentally delayed 2nd-instars, followed by eventually succumbing to nonhost resistance defense mechanisms.

Insect derived extra oral GH32 plays a role in susceptibility of wheat to Hessian fly.

The Hessian fly is an obligate parasite of wheat causing significant economic damage, and triggers either a resistant or susceptible reaction. However, the molecular mechanisms of susceptibility leading to the establishment of the larvae are unknown. Larval survival on the plant requires the establishment of a steady source of readily available nutrition. Unlike other insect pests, the Hessian fly larvae have minute mandibles and cannot derive their nutrition by chewing tissue or sucking phloem sap. Here, we show that the virulent larvae produce the glycoside hydrolase MdesGH32 extra-orally, that localizes within the leaf tissue being fed upon. MdesGH32 has strong inulinase and invertase activity aiding in the breakdown of the plant cell wall inulin polymer into monomers and converting sucrose, the primary transport sugar in plants, to glucose and fructose, resulting in the formation of a nutrient-rich tissue. Our finding elucidates the molecular mechanism of nutrient sink formation and establishment of susceptibility.

Phenotypic and molecular characterization of Hessian fly resistance in diploid wheat, Aegilops tauschii.

BACKGROUND: The Hessian fly (Mayetiola destructor), belonging to the gall midge family (Cecidomyiidae), is a devastating pest of wheat (Triticum aestivum) causing significant yield losses. Despite identification and characterization of numerous Hessian fly-responsive genes and associated biological pathways involved in wheat defense against this dipteran pest, their functional validation has been challenging. This is largely attributed to the large genome, polyploidy, repetitive DNA, and limited genetic resources in hexaploid wheat. The diploid progenitor Aegilops tauschii, D-genome donor of modern-day hexaploid wheat, offers an ideal surrogate eliminating the need to target all three homeologous chromosomes (A, B and D) individually, and thereby making the functional validation of candidate Hessian fly-responsive genes plausible. Furthermore, the well-annotated sequence of Ae. tauschii genome and availability of genetic resources amenable to manipulations makes the functional assays less tedious and time-consuming. However, prior to utilization of this diploid genome for downstream studies, it is imperative to characterize its physical and molecular responses to Hessian fly. RESULTS: In this study we screened five Ae. tauschii accessions for their response to the Hessian fly biotypes L and vH13. Two lines were identified that exhibited a homozygous resistance response to feeding by both Hessian fly biotypes. Studies using physical measurements and neutral red staining showed that the resistant Ae. tauschii accessions resembled hexaploid wheat in their phenotypic responses to Hessian fly, that included similarities in larval developmental stages, leaf and plant growth, and cell wall permeability. Furthermore, molecular responses, characterized by gene expression profiling using quantitative real-time PCR, in select resistant Ae. tauschii lines also revealed similarities with resistant hexaploid wheat. CONCLUSIONS: Phenotypic and molecular characterization of Ae. tauschii to Hessian fly infestation revealed resistant accessions that shared similarities to hexaploid wheat. Resembling the resistant hexaploid wheat, the Ae. tauschii accessions mount an early defense strategy involving defense proteins including lectins, secondary metabolites and reactive oxygen species (ROS) radicals. Our results reveal the suitability of the diploid progenitor for use as an ideal tool for functional genomics research in deciphering the wheat-Hessian fly molecular interactions.

Multiple molecular defense strategies in Brachypodium distachyon surmount Hessian fly (Mayetiola destructor) larvae-induced susceptibility for plant survival.

The Hessian fly is a destructive pest of wheat causing severe economic damage. Numerous genes and associated biological pathways have been implicated in defense against Hessian fly. However, due to limited genetic resources, compounded with genome complexity, functional analysis of the candidate genes are challenging in wheat. Physically, Brachypodium distachyon (Bd) exhibits nonhost resistance to Hessian fly, and with a small genome size, short life cycle, vast genetic resources and amenability to transformation, it offers an alternate functional genomic model for deciphering plant-Hessian fly interactions. RNA-sequencing was used to reveal thousands of Hessian fly-responsive genes in Bd one, three, and five days after egg hatch. Genes encoding defense proteins, stress-regulating transcription factors, signaling kinases, and secondary metabolites were strongly up-regulated within the first 24 hours of larval feeding indicating an early defense, similar to resistant wheat. Defense was mediated by a hypersensitive response that included necrotic lesions, up-regulated ROS-generating and -scavenging enzymes, and H2O2 production. Suppression of cell wall-associated proteins and increased cell permeability in Bd resembled susceptible wheat. Thus, Bd molecular responses shared similarities to both resistant and susceptible wheat, validating its suitability as a model genome for undertaking functional studies of candidate Hessian fly-responsive genes.

Modulation of nonessential amino acid biosynthetic pathways in virulent Hessian fly larvae (Mayetiola destructor), feeding on susceptible host wheat (Triticum aestivum).

Compatible interactions between wheat (Triticum aestivum), and its dipteran pest Hessian fly (Hf, Mayetiola destructor) result in successful establishment of larval feeding sites rendering the host plant susceptible. Virulent larvae employ an effector-based feeding strategy to reprogram the host physiology resulting in formation of a protein- and sugar-rich nutritive tissue beneficial to developing larvae. Previous studies documented increased levels of nonessential amino acids (NAA; that need not be received through insect diet) in the susceptible wheat in response to larval feeding, suggesting importance of plant-derived NAA in larval nutrition. Here, we investigated the modulation of genes from NAA biosynthetic pathways (NAABP) in virulent Hf larvae. Transcript profiling for 16 NAABP genes, annotated from the recently assembled Hf genome, was carried out in the feeding first-, and second-instars and compared with that of the first-instar neonate (newly hatched, migrating, assumed to be non-feeding) larvae. While Tyr, Gln, Glu, and Pro NAABP genes transcript abundance declined in the feeding instars as compared to the neonates, those for Ala, and Ser increased in the feeding larval instars, despite higher levels of these NAA in the susceptible host plant. Asp, Asn, Gly and Cys NAABP genes exhibited variable expression profiles in the feeding first- and second-instars. Our results indicate that while Hf larvae utilize the plant-derived NAA, de novo synthesis of several NAA may be necessary to: (i) provide larvae with the requisite amount for sustaining growth before nutritive tissue formation and, (ii) overcome any inadequate amounts in the host plant, post-nutritive tissue formation.

Induced epidermal permeability modulates resistance and susceptibility of wheat seedlings to herbivory by Hessian fly larvae.

Salivary secretions of neonate Hessian fly larvae initiate a two-way exchange of molecules with their wheat host. Changes in properties of the leaf surface allow larval effectors to enter the plant where they trigger plant processes leading to resistance and delivery of defence molecules, or susceptibility and delivery of nutrients. To increase understanding of the host plant's response, the timing and characteristics of the induced epidermal permeability were investigated. Resistant plant permeability was transient and limited in area, persisting just long enough to deliver defence molecules before gene expression and permeability reverted to pre-infestation levels. The abundance of transcripts for GDSL-motif lipase/hydrolase, thought to contribute to cuticle reorganization and increased permeability, followed the same temporal profile as permeability in resistant plants. In contrast, susceptible plants continued to increase in permeability over time until the entire crown of the plant became a nutrient sink. Permeability increased with higher infestation levels in susceptible but not in resistant plants. The ramifications of induced plant permeability on Hessian fly populations are discussed.

Changes in properties of wheat leaf cuticle during interactions with Hessian fly.

Infestation of wheat by Hessian fly larvae causes a variety of physical and biochemical modifications of the host plant. Changes occur in cuticle permeability, lipid composition and gene transcript abundance, and these responses differ substantially between resistant and susceptible wheat lines. Staining assays revealed that susceptible plants exhibited a generalized increase in leaf sheath epidermal permeability during infestation; whereas, epidermal permeability was only minimally affected in resistant plants. Furthermore, temporal profiling using gas chromatographic methods revealed that changes in cuticle lipid (wax and cutin) composition correlated well with differing levels of epidermal permeability in susceptible and resistant plants. Temporal analysis of cuticle-associated gene mRNA levels, by quantitative real-time PCR, indicated a relationship between transcript abundance and changes in cuticle lipid profiles of resistant and susceptible plants. Results suggest that conserving cuticle integrity via induction of specific wax constituents and maintenance of cutin amounts, determined by the accumulation of cuticle-associated transcripts, could be important components of wheat resistance to Hessian fly larvae.

Reactive oxygen species are involved in plant defense against a gall midge.

Reactive oxygen species (ROS) play a major role in plant defense against pathogens, but evidence for their role in defense against insects is still preliminary and inconsistent. In this study, we examined the potential role of ROS in defense of wheat (Triticum aestivum) and rice (Oryza sativa) against Hessian fly (Mayetiola destructor) larvae. Rapid and prolonged accumulation of hydrogen peroxide (H(2)O(2)) was detected in wheat plants at the attack site during incompatible interactions. Increased accumulation of both H(2)O(2) and superoxide was detected in rice plants during nonhost interactions with the larvae. No increase in accumulation of either H(2)O(2) or superoxide was observed in wheat plants during compatible interactions. A global analysis revealed changes in the abundances of 250 wheat transcripts and 320 rice transcripts encoding proteins potentially involved in ROS homeostasis. A large number of transcripts encoded class III peroxidases that increased in abundance during both incompatible and nonhost interactions, whereas the levels of these transcripts decreased in susceptible wheat during compatible interactions. The higher levels of class III peroxidase transcripts were associated with elevated enzymatic activity of peroxidases at the attack site in plants during incompatible and nonhost interactions. Overall, our data indicate that class III peroxidases may play a role in ROS generation in resistant wheat and nonhost rice plants during response to Hessian fly attacks.

Gene-for-gene defense of wheat against the Hessian fly lacks a classical oxidative burst.

Genetic similarities between plant interactions with microbial pathogens and wheat interactions with Hessian fly larvae prompted us to investigate defense and counterdefense mechanisms. Plant oxidative burst, a rapid increase in the levels of active oxygen species (AOS) within the initial 24 h of an interaction with pathogens, commonly is associated with defenses that are triggered by gene-for-gene recognition events similar to those involving wheat and Hessian fly larvae. RNAs encoded by Hessian fly superoxide dismutase (SOD) and catalase (CAT) genes, involved in detoxification of AOS, increased in first-instar larvae during both compatible and incompatible interactions. However, mRNA levels of a wheat NADPH oxidase (NOX) gene that generates superoxide (O2-) did not increase. In addition, inhibiting wheat NOX enzyme with diphenyleneiodonium did not result in increased survival of avirulent larvae. However, nitro blue tetrazolium staining indicated that basal levels of O2- are present in both uninfested and infested wheat tissue. mRNA encoded by wheat genes involved in detoxification of the cellular environment, SOD, CAT, and glutathione-S-transferase did not increase in abundance. Histochemical staining with 3,3-diaminobenzidine revealed no increases in wheat hydrogen peroxide (H2O2) during infestation that were correlated with the changes in larval SOD and CAT mRNA. However, treatment with 2',7'-dichlorofluorescin demonstrated the presence of basal levels of H2O2 in the elongation zone of both infested and uninfested plants. The accumulation of a wheat flavanone 3-hydroxylase mRNA did show some parallels with larval gene mRNA profiles. These results suggested that larvae encounter stresses imposed by mechanisms other than an oxidative burst in wheat seedlings.

Hfr-2, a wheat cytolytic toxin-like gene, is up-regulated by virulent Hessian fly larval feedingdouble dagger.

SUMMARY Both yield and grain-quality are dramatically decreased when susceptible wheat (Triticum aestivum) plants are infested by Hessian fly (Mayetiola destructor) larvae. Examination of the changes in wheat gene expression during infestation by virulent Hessian fly larvae has identified the up-regulation of a gene, Hessian fly responsive-2 (Hfr-2), which contains regions similar to genes encoding seed-specific agglutinin proteins from Amaranthus. Hfr-2, however, did not accumulate in developing seeds, as do other wheat seed storage proteins. Additionally, a separate region of the HFR-2 predicted amino acid sequence is similar to haemolytic proteins, from both mushroom and bacteria, that are able to form pores in cell membranes of mammalian red blood cells. The involvement of Hfr-2 in interactions with insects was supported by experiments demonstrating its up-regulation by both fall armyworm (Spodoptera frugiperda) and bird cherry-oat aphid (Rhopalosiphum padi) infestations but not by virus infection. Examination of wheat defence response pathways showed Hfr-2 up-regulation following methyl jasmonate treatment and only slight up-regulation in response to salicylic acid, abscisic acid and wounding treatments. Like related proteins, HFR-2 may normally function in defence against certain insects or pathogens. However, we propose that as virulent Hessian fly larvae manipulate the physiology of the susceptible host, the HFR-2 protein inserts in plant cell membranes at the feeding sites and by forming pores provides water, ions and other small nutritive molecules to the developing larvae.

A lectin-like wheat gene responds systemically to attempted feeding by avirulent first-instar Hessian fly larvae.

Through gene-for-gene interactions, wheat plants respond to specific biotypes of Hessian fly upon the initiation of first-instar larval feeding. Leaves of plants containing the H9 resistance gene responded to avirulent biotype L. larvae with rapid changes in the levels of several mRNA transcripts and initiation of an incompatible interaction. A low-copy gene, Hfr-1 (Hessian fly-response gene 1), responded with increased mRNA levels for two days before returning to preinfestation levels by day five. Hfr-1 mRNA was constitutively expressed in uninfested control plants as well as in plants infested with virulent larvae. The cDNA sequence was similar to a maize gene encoding a beta-glucosidase aggregating factor (BGAF), to jacalin-like mannose-binding lectins, and to several plant genes that respond to microbial infections. The potential roles of Hfr-1 in defending wheat against Hessian fly damage are discussed.