Untapped Sources of Dual Resistance to Hessian Fly and Greenbug in Synthetic Hexaploid Wheats.

The Hessian fly (Hf) and greenbugs (Gb) are major pests of wheat, causing severe economic losses globally. Deploying resistant wheat is the most effective strategy for managing these destructive insects. However, the resistance is not effective against all Hf or Gb biotypes and can impose selection pressure on insects, resulting in the development of virulent biotypes. These challenges must be met through the discovery of new and novel sources of resistance to these pests. Synthetic Hexaploid Wheat (SHW)-developed cultivars are a rich source of resistance against a diverse array of pathogens and pests. In this study, 80 SHW lines were evaluated for their resistance to Hf and Gb under controlled environmental conditions. Of these, a total of 36 SHW lines showed resistance independently to Hf biotype L and Gb biotype E, while 27 lines showed combined resistance to both Hf and Gb. Further, a subset of 10 SHW lines showed resistance to additional Hf biotypes, Great Plains and vH13. The identification of SHW lines resistant to multiple insects and biotypes offers an invaluable resource to breeders who are looking to stack resistance traits to develop elite cultivars as a strategy to alleviate economic impacts upon global wheat production.

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum.

Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.

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.

Improved soybean transformation for efficient and high throughput transgenic production.

Although genetic transformation of soybean dates back to over two decades, the process remains inefficient. Here, we report the development of an organogenesis-based transformation method of soybean that resulted in an average transformation frequency of 18.7%. This improved method resorts to Agrobacterium-mediated transformation of the split-seed explant with an attached partial embryonic axis obtained from an imbibed seed. In addition to the split-seed explant, Agrobacterium strain and preparation were shown to be important for improved transformation. Transformation with Agrobacterium tumefaciens EHA105 generated higher transformation frequencies and number of low copy events compared to the strain EHA101. In this system, phosphinothricin acetyl transferase conferring tolerance to glufosinate was successfully employed for efficiently producing transgenic events. Around 48% of the T1 progeny was demonstrated to be heritable based on molecular analysis and screening with the herbicide Liberty®. This method was shown to be applicable to different genotypes and a few elite lines showed high transformation frequencies. This split-seed system with an attached partial embryonic axis serves not only as an efficient means for high throughput transgenic production for basic research studies but also for the commercial development of transgenic soybean products.

Using Morphogenic Genes to Improve Recovery and Regeneration of Transgenic Plants.

Efficient transformation of numerous important crops remains a challenge, due predominantly to our inability to stimulate growth of transgenic cells capable of producing plants. For years, this difficulty has been partially addressed by tissue culture strategies that improve regeneration either through somatic embryogenesis or meristem formation. Identification of genes involved in these developmental processes, designated here as morphogenic genes, provides useful tools in transformation research. In species from eudicots and cereals to gymnosperms, ectopic overexpression of genes involved in either embryo or meristem development has been used to stimulate growth of transgenic plants. However, many of these genes produce pleiotropic deleterious phenotypes. To mitigate this, research has been focusing on ways to take advantage of growth-stimulating morphogenic genes while later restricting or eliminating their expression in the plant. Methods of controlling ectopic overexpression include the use of transient expression, inducible promoters, tissue-specific promoters, and excision of the morphogenic genes. These methods of controlling morphogenic gene expression have been demonstrated in a variety of important crops. Here, we provide a review that highlights how ectopic overexpression of genes involved in morphogenesis has been used to improve transformation efficiencies, which is facilitating transformation of numerous recalcitrant crops. The use of morphogenic genes may help to alleviate one of the bottlenecks currently slowing progress in plant genome modification.

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.

Zinc finger nuclease-mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non-homologous end joining.

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence-specific endonucleases to generate DNA double strand breaks (DSBs) at user-specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non-homologous end joining (NHEJ)-mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology-directed repair (HDR)-mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2-1a (FAD2-1a) locus of embryogenic cells in tissue culture. We then describe ZFN- and NHEJ-mediated, targeted integration of two different multigene donors to the FAD2-1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T1 progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ-integrated donor were perfect or near-perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ-mediated targeted insertions of multigene donors at an endogenous genomic locus.

Coexpression of octopine and succinamopine Agrobacterium virulence genes to generate high quality transgenic events in maize by reducing vector backbone integration.

Agrobacterium-mediated transformation is a complex process that is widely utilized for generating transgenic plants. However, one of the major concerns of this process is the frequent presence of undesirable T-DNA vector backbone sequences in the transgenic plants. To mitigate this deficiency, a ternary strain of A. tumefaciens was modified to increase the precision of T-DNA border nicking such that the backbone transfer is minimized. This particular strain supplemented the native succinamopine VirD1/VirD2 of EHA105 with VirD1/VirD2 derived from an octopine source (pTi15955), the same source as the binary T-DNA borders tested here, residing on a ternary helper plasmid containing an extra copy of the succinamopine VirB/C/G operons and VirD1. Transformation of maize immature embryos was carried out with two different test constructs, pDAB101556 and pDAB111437, bearing the reporter YFP gene and insecticidal toxin Cry1Fa gene, respectively, contained in the VirD-supplemented and regular control ternary strains. Molecular analyses of ~ 700 transgenic events revealed a significant 2.6-fold decrease in events containing vector backbone sequences, from 35.7% with the control to 13.9% with the VirD-supplemented strain for pDAB101556 and from 24.9% with the control to 9.3% with the VirD-supplemented strain for pDAB111437, without compromising transformation efficiency. In addition, while the number of single copy events recovered was similar, there was a 24-26% increase in backbone-free events with the VirD-supplemented strain compared to the control strain. Thus, supplementing existing VirD1/VirD2 genes in Agrobacterium, to recognize diverse T-DNA borders, proved to be a useful tool to increase the number of high quality events in maize.

Correction to: Agrobacterium: A Genome-Editing Tool-Delivery System.

By mistake the chapter was published with incorrect author name. The chapter has now been corrected.

Agrobacterium: A Genome-Editing Tool-Delivery System.

With the rapidly increasing global population, it will be extremely challenging to provide food to the world without increasing food production by at least 70% over the next 30 years. As we reach the limits of expanding arable land, the responsibility of meeting this production goal will rely on increasing yields. Traditional plant breeding practices will not be able to realistically meet these expectations, thrusting plant biotechnology into the limelight to fulfill these needs. Better varieties will need to be developed faster and with the least amount of regulatory hurdles. With the need to add, delete, and substitute genes into existing genomes, the field of genome editing and gene targeting is now rapidly developing with numerous new technologies coming to the forefront. Agrobacterium-mediated crop transformation has been the most utilized method to generate transgenic varieties that are better yielding, have new traits, and are disease and pathogen resistant. Genome-editing technologies rely on the creation of double-strand breaks (DSBs) in the genomic DNA of target species to facilitate gene disruption, addition, or replacement through either non-homologous end joining or homology-dependent repair mechanisms. DSBs can be introduced through the use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspersed short palindromic repeats (CRISPR)/Cas nucleases, among others. Agrobacterium strains have been employed to deliver the reagents for genome editing to the specific target cells. Understanding the biology of transformation from the perspective not only of Agrobacterium, but also of the host, from processing of T-DNA to its integration in the host genome, has resulted in a wealth of information that has been used to engineer Agrobacterium strains having increased virulence. As more technologies are being developed, that will help overcome issues of Agrobacterium host range and random integration of DNA, combined with highly sequence-specific nucleases, a robust crop genome-editing toolkit finally seems attainable.

Comparison of the impact of viral and plant-derived promoters regulating selectable marker gene on maize transformation and transgene expression.

KEY MESSAGE: The choice of promoter regulating the selectable marker gene impacts transformation efficiency, copy number and the expression of selectable marker and flanking genes in maize. Viral or plant-derived constitutive promoters are often used to regulate selectable marker genes. We compared two viral promoters, cauliflower mosaic virus (CaMV 35T) and sugarcane bacilliform virus (SCBV) with two plant promoters, rice actin1 (OsAct1) and maize ubiquitin 1 (ZmUbi1) to drive aryloxyalkanoate dioxygenase (aad-1) selectable marker gene in maize inbred line B104. ZmUbi1- and OsAct1-containing constructs demonstrated higher transformation frequencies (43.8 and 41.4%, respectively) than the two viral promoter constructs, CaMV 35T (25%) and SCBV (8%). Interestingly, a higher percentage of single copy events were recovered for SCBV (82.1%) and CaMV 35T (59.3%) promoter constructs, compared to the two plant-derived promoters, OsAct1 (40.0%), and ZmUbi1 (27.6%). Analysis of protein expression suggested that the viral promoter CaMV 35T expressed significantly higher AAD-1 protein (174.6 ng/cm2) than the OsAct1 promoter (12.6 ng/cm2) in T0 leaf tissue. When measured in T2 callus tissue, the two viral promoters both had higher expression and more variability than the two plant-derived promoters. A potential explanation for why viral promoters produce lower transformation efficiencies but higher percentages of low copy number events is discussed. In addition, viral promoters regulating aad-1 were found to influence the expression of upstream flanking genes in both T0 leaf and T2 callus tissue.

Hessian fly larval feeding triggers enhanced polyamine levels in susceptible but not resistant wheat.

BACKGROUND: Hessian fly (Mayetiola destructor), a member of the gall midge family, is one of the most destructive pests of wheat (Triticum aestivum) worldwide. Probing of wheat plants by the larvae results in either an incompatible (avirulent larvae, resistant plant) or a compatible (virulent larvae, susceptible plant) interaction. Virulent larvae induce the formation of a nutritive tissue, resembling the inside surface of a gall, in susceptible wheat. These nutritive cells are a rich source of proteins and sugars that sustain the developing virulent Hessian fly larvae. In addition, on susceptible wheat, larvae trigger a significant increase in levels of amino acids including proline and glutamic acid, which are precursors for the biosynthesis of ornithine and arginine that in turn enter the pathway for polyamine biosynthesis. RESULTS: Following Hessian fly larval attack, transcript abundance in susceptible wheat increased for several genes involved in polyamine biosynthesis, leading to higher levels of the free polyamines, putrescine, spermidine and spermine. A concurrent increase in polyamine levels occurred in the virulent larvae despite a decrease in abundance of Mdes-odc (ornithine decarboxylase) transcript encoding a key enzyme in insect putrescine biosynthesis. In contrast, resistant wheat and avirulent Hessian fly larvae did not exhibit significant changes in transcript abundance of genes involved in polyamine biosynthesis or in free polyamine levels. CONCLUSIONS: The major findings from this study are: (i) although polyamines contribute to defense in some plant-pathogen interactions, their production is induced in susceptible wheat during interactions with Hessian fly larvae without contributing to defense, and (ii) due to low abundance of transcripts encoding the rate-limiting ornithine decarboxylase enzyme in the larval polyamine pathway the source of polyamines found in virulent larvae is plausibly wheat-derived. The activation of the host polyamine biosynthesis pathway during compatible wheat-Hessian fly interactions is consistent with a model wherein the virulent larvae usurp the polyamine biosynthesis machinery of the susceptible plant to acquire nutrients required for their own growth and development.

The Arabidopsis Myb transcription factor MTF1 is a unidirectional regulator of susceptibility to Agrobacterium.

We recently described the Arabidopsis Myb transcription factor MTF1 that negatively regulates plant susceptibility to Agrobacterium-mediated transformation. Roots of mtf1 mutant plants show increased susceptibility to several Agrobacterium strains, and complementing the mutants with a MTF1 cDNA decreases transformation susceptibility to wild-type levels. Here, we show that overexpression of MTF1 in a wild-type Arabidopsis background does not result in altered transformation susceptibility. However, MTF1 overexpressing plants show increased root length and larger and darker leaves, indicating that MTF1 plays a role in plant growth and development. MTF1 decreases Arabidopsis root susceptibility specifically to Agrobacterium but plant responses to the pathogens Alternaria brassicicola or Pseudomonas syringae pv Tomato were not altered. However, the homozygous MTF1 mutant mtf1-4 is resistant to Botrytis cinerea strain BO5-10 and is regulated through the ethylene signaling pathway mediated by upregulation of the AP2/ERF transcription factor ORA59.

Cytokinins secreted by Agrobacterium promote transformation by repressing a plant myb transcription factor.

Agrobacterium-mediated transformation is the most widely used technique for generating transgenic plants. However, many crops remain recalcitrant. We found that an Arabidopsis myb family transcription factor (MTF1) inhibited plant transformation susceptibility. Mutating MTF1 increased attachment of several Agrobacterium strains to roots and increased both stable and transient transformation in both susceptible and transformation-resistant Arabidopsis ecotypes. Cytokinins from Agrobacterium tumefaciens decreased the expression of MTF1 through activation of the cytokinin response regulator ARR3. Mutating AHK3 and AHK4, genes that encode cytokinin-responsive kinases, increased the expression of MTF1 and impaired plant transformation. Mutant mtf1 plants also had increased expression of AT14A, which encodes a putative transmembrane receptor for cell adhesion molecules. Plants overexpressing AT14A exhibited increased susceptibility to transformation, whereas at14a mutant plants exhibited decreased attachment of bacteria to roots and decreased transformation, suggesting that AT14A may serve as an anchor point for Agrobacteria. Thus, by promoting bacterial attachment and transformation of resistant plants and increasing such processes in susceptible plants, treating roots with cytokinins may help engineer crops with improved features or yield.

Functional characterization of HFR1, a high-mannose N-glycan-specific wheat lectin induced by Hessian fly larvae.

We previously cloned and characterized a novel jacalin-like lectin gene from wheat (Triticum aestivum) plants that responds to infestation by Hessian fly (Mayetiola destructor) larvae, a major dipteran pest of this crop. The infested resistant plants accumulated higher levels of Hfr-1 (for Hessian fly-responsive gene 1) transcripts compared with uninfested or susceptible plants. Here, we characterize the soluble and active recombinant His(6)-HFR1 protein isolated from Escherichia coli. Functional characterization of the protein using hemagglutination assays revealed lectin activity. Glycan microarray-binding assays indicated strong affinity of His(6)-HFR1 to Manalpha1-6(Manalpha1-3)Man trisaccharide structures. Resistant wheat plants accumulated high levels of HFR1 at the larval feeding sites, as revealed by immunodetection, but the avirulent larvae were deterred from feeding and consumed only small amounts of the lectin. Behavioral studies revealed that avirulent Hessian fly larvae on resistant plants exhibited prolonged searching and writhing behaviors as they unsuccessfully attempted to establish feeding sites. During His(6)-HFR1 feeding bioassays, Drosophila melanogaster larvae experienced significant delays in growth and pupation, while percentage mortality increased with progressively higher concentrations of His(6)-HFR1 in the diet. Thus, HFR1 is an antinutrient to dipteran larvae and may play a significant role in deterring Hessian fly larvae from feeding on resistant wheat plants.

cDNA cloning and transcriptional expression of a peritrophin-like gene in the Hessian fly, Mayetiola destructor [Say].

One of the well-studied components of the insect gut is the peritrophic matrix (PM). This semipermeable structure primarily functions in digestion, and protection against invasive microorganisms and mechanical damage. We report the cDNA cloning and transcription profiles of a peritrophin-A like gene (designated MdesPERI-A1) in the Hessian fly Mayetiola destructor. The predicted amino acid sequence of MdesPERI-A1 revealed a putative secretion signal peptide at its amino terminus, similarity to peritrophins from other insects including dipterans, and the presence of two chitin binding domains each containing six cysteine residues. Quantitative expression analysis of MdesPERI-A1 mRNA in different larval tissues revealed the transcript to be predominantly present in the midgut (597.9-fold) compared to other tissues assayed including salivary glands and fat bodies. Spatial expression patterns during development showed a peak expression of MdesPERI-A1 in the feeding second-instars (146-fold) and a decline in expression in the pupal and adult stages. Transcription profiling of MdesPERI-A1 during compatible (larvae on susceptible plants) and incompatible (larvae on resistant plants) interactions with wheat revealed a greater level (1.7-fold) of MdesPERI-A1 transcript in larvae on resistant plants in the initial time point examined. However, MdesPERI-A1 expression declined in larvae on resistant plants at the later time points.

Behavioral genomics of honeybee foraging and nest defense.

The honeybee has been the most important insect species for study of social behavior. The recently released draft genomic sequence for the bee will accelerate honeybee behavioral genetics. Although we lack sufficient tools to manipulate this genome easily, quantitative trait loci (QTLs) that influence natural variation in behavior have been identified and tested for their effects on correlated behavioral traits. We review what is known about the genetics and physiology of two behavioral traits in honeybees, foraging specialization (pollen versus nectar), and defensive behavior, and present evidence that map-based cloning of genes is more feasible in the bee than in other metazoans. We also present bioinformatic analyses of candidate genes within QTL confidence intervals (CIs). The high recombination rate of the bee made it possible to narrow the search to regions containing only 17-61 predicted peptides for each QTL, although CIs covered large genetic distances. Knowledge of correlated behavioral traits, comparative bioinformatics, and expression assays facilitated evaluation of candidate genes. An overrepresentation of genes involved in ovarian development and insulin-like signaling components within pollen foraging QTL regions suggests that an ancestral reproductive gene network was co-opted during the evolution of foraging specialization. The major QTL influencing defensive/aggressive behavior contains orthologs of genes involved in central nervous system activity and neurogenesis. Candidates at the other two defensive-behavior QTLs include modulators of sensory signaling (Am5HT(7) serotonin receptor, AmArr4 arrestin, and GABA-B-R1 receptor). These studies are the first step in linking natural variation in honeybee social behavior to the identification of underlying genes.

Expression patterns of antibacterial genes in the Hessian fly.

We report on the transcriptional patterns of three antibacterial genes, a defensin (MdesDEF-1), a diptericin (MdesDIP-1) and a lysozyme (MdesLYS-1), during development in Hessian fly, Mayetiola destructor. Quantitative analysis by real-time PCR of mRNA levels in different tissues revealed a predominance of the transcripts for all three genes in the midgut, while analysis during development revealed greatest abundance in mRNA during the 3rd-instar. An evaluation of the midgut lumen revealed the presence of a diverse bacterial flora in larvae maintained on susceptible wheat. Further, the titer of bacteria in the midgut increased approximately 250-fold from the 1st-instar through the 2nd-instar. However, no detectable titer of bacteria was observed from the midgut lumen of larvae maintained on resistant plants. PCR amplicons produced using primers designed to conserved regions of the Pseudomonas 16S rRNA gene supported taxonomic identification for some of the bacteria comprising the midgut flora as belonging to the genus Pseudomonas. Analysis of mRNA for the Hessian fly antibacterial genes in larvae feeding on susceptible and resistant plants revealed an increase in the transcript level for MdesDEF-1 in 1st-instar larvae on susceptible plants, while the transcript levels for MdesDIP-1 and MdesLYS-1 were constant. Results suggest the transcriptional patterns of the Hessian fly antibacterial genes observed could be associated with the developing midgut bacterial flora present in larvae feeding on susceptible wheat as well as microbial challenge encountered at other stages in development.

Constitutive expression exposes functional redundancy between the Arabidopsis histone H2A gene HTA1 and other H2A gene family members.

The Arabidopsis thaliana histone H2A gene HTA1 is essential for efficient transformation of Arabidopsis roots by Agrobacterium tumefaciens. Disruption of this gene in the rat5 mutant results in decreased transformation. In Arabidopsis, histone H2A proteins are encoded by a 13-member gene family. RNA encoded by these genes accumulates to differing levels in roots and whole plants; HTA1 transcripts accumulate to levels up to 1000-fold lower than do transcripts of other HTA genes. We examined the extent to which other HTA genes or cDNAs could compensate for loss of HTA1 activity when overexpressed in rat5 mutant plants. Overexpression of all tested HTA cDNAs restored transformation competence to the rat5 mutant. However, only the HTA1 gene, but not other HTA genes, could phenotypically complement rat5 mutant plants when expressed from their native promoters. Expression analysis of HTA promoters indicated that they had distinct but somewhat overlapping patterns of expression in mature plants. However, only the HTA1 promoter was induced by wounding or by Agrobacterium infection of root segments. Our data suggest that, with respect to Agrobacterium-mediated transformation, all tested histone H2A proteins are functionally redundant. However, this functional redundancy is not normally evidenced because of the different expression patterns of the HTA genes.