BED domain-containing NLR from wild barley confers resistance to leaf rust.

Leaf rust, caused by Puccinia hordei, is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Despite the effectiveness of genetic resistance, the deployment of single genes often compromises durability due to the emergence of virulent P. hordei races, prompting the search for new sources of resistance. Here we report on the cloning of Rph15, a resistance gene derived from barley's wild progenitor H. vulgare subsp. spontaneum. We demonstrate using introgression mapping, mutation and complementation that the Rph15 gene from the near-isogenic line (NIL) Bowman + Rph15 (referred to as BW719) encodes a coiled-coil nucleotide-binding leucine-rich repeat (NLR) protein with an integrated Zinc finger BED (ZF-BED) domain. A predicted KASP marker was developed and validated across a collection of Australian cultivars and a series of introgression lines in the Bowman background known to carry the Rph15 resistance. Rph16 from HS-680, another wild barley derived leaf rust resistance gene, was previously mapped to the same genomic region on chromosome 2H and was assumed to be allelic with Rph15 based on genetic studies. Both sequence analysis, race specificity and the identification of a knockout mutant in the HS-680 background suggest that Rph15- and Rph16-mediated resistances are in fact the same and not allelic as previously thought. The cloning of Rph15 now permits efficient gene deployment and the production of resistance gene cassettes for sustained leaf rust control.

A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in wheat.

Breeding wheat with durable resistance to the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), a major threat to cereal production, is challenging due to the rapid evolution of pathogen virulence. Increased durability and broad-spectrum resistance can be achieved by introducing more than one resistance gene, but combining numerous unlinked genes by breeding is laborious. Here we generate polygenic Pgt resistance by introducing a transgene cassette of five resistance genes into bread wheat as a single locus and show that at least four of the five genes are functional. These wheat lines are resistant to aggressive and highly virulent Pgt isolates from around the world and show very high levels of resistance in the field. The simple monogenic inheritance of this multigene locus greatly simplifies its use in breeding. However, a new Pgt isolate with virulence to several genes at this locus suggests gene stacks will need strategic deployment to maintain their effectiveness.

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley.

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley production in Africa and Europe. In contrast to wheat with 60 designated stem rust (Sr) resistance genes, barley's genetic variation for stem rust resistance is very narrow with only ten resistance genes genetically identified. Of these, only one complex locus consisting of three genes is effective against TTKSK, a widely virulent Pgt race of the Ug99 tribe which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against race TTKSK. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Furthermore, these transgenic plants did not display significant adverse agronomic effects in the absence of disease. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley.

Extensive Genetic Variation at the Sr22 Wheat Stem Rust Resistance Gene Locus in the Grasses Revealed Through Evolutionary Genomics and Functional Analyses.

In the last 20 years, severe wheat stem rust outbreaks have been recorded in Africa, Europe, and Central Asia. This previously well controlled disease, caused by the fungus Puccinia graminis f. sp. tritici, has reemerged as a major threat to wheat cultivation. The stem rust (Sr) resistance gene Sr22 encodes a nucleotide-binding and leucine-rich repeat receptor which confers resistance to the highly virulent African stem rust isolate Ug99. Here, we show that the Sr22 gene is conserved among grasses in the Triticeae and Poeae lineages. Triticeae species contain syntenic loci with single-copy orthologs of Sr22 on chromosome 7, except Hordeum vulgare, which has experienced major expansions and rearrangements at the locus. We also describe 14 Sr22 sequence variants obtained from both Triticum boeoticum and the domesticated form of this species, T. monococcum, which have been postulated to encode both functional and nonfunctional Sr22 alleles. The nucleotide sequence analysis of these alleles identified historical sequence exchange resulting from recombination or gene conversion, including breakpoints within codons, which expanded the coding potential at these positions by introduction of nonsynonymous substitutions. Three Sr22 alleles were transformed into wheat cultivar Fielder and two postulated resistant alleles from Schomburgk (hexaploid wheat introgressed with T. boeoticum segment carrying Sr22) and T. monococcum accession PI190945, respectively, conferred resistance to P. graminis f. sp. tritici race TTKSK, thereby unequivocally confirming Sr22 effectiveness against Ug99. The third allele from accession PI573523, previously believed to confer susceptibility, was confirmed as nonfunctional against Australian P. graminis f. sp. tritici race 98-1,2,3,5,6.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abiotic stress upregulated TaZFP34 represses the expression of type-B response regulator and SHY2 genes and enhances root to shoot ratio in wheat.

Q-type C2H2 zinc finger proteins (ZFPs) are plant-specific DNA-binding proteins containing a conserved QALGGH motif. This study investigated the function of abiotic stress-inducible and predominantly root-expressed Triticum aestivum ZFPs (TaZFP22, TaZFP34 and TaZFP46) with a focus on TaZFP34. Expression of TaZFP34 in roots was upregulated by high salinity, dehydration, oxidative and cold stresses. Overexpression of TaZFP34 in wheat roots resulted in an increased root-to-shoot ratio, a phenomenon observed during plant adaptation to drying soil. Expression of a number of genes which are potentially involved in modulating root growth was significantly altered in the roots of TaZFP34 overexpressing lines. In particular, the transcript levels of TaRR12B, TaRR12D and TaSHY2 that are homologues of known negative regulators of root growth were significantly reduced. Expression of shoot growth-related genes, such as GA3-ox and expansins, was downregulated in the transgenic shoots. TaZFP34 bound to (C/G)AGT(G/A)-like elements in the promoters of TaZFP34 down-regulated TaRR12D and TaSHY2 and transrepressed the reporter gene expression driven by TaRR12D and TaSHY2 promoters. Expression of the above reporter genes was also repressed by TaZFP46 and TaZFP22. These data suggest that TaZFP34 is a transcriptional repressor and is involved in modulating the root-to-shoot ratio.

Drought-Up-Regulated TaNAC69-1 is a Transcriptional Repressor of TaSHY2 and TaIAA7, and Enhances Root Length and Biomass in Wheat.

A well-known physiological adaptation process of plants encountering drying soil is to achieve water balance by reducing shoot growth and maintaining or promoting root elongation, but little is known about the molecular basis of this process. This study investigated the role of a drought-up-regulated Triticum aestivum NAC69-1 (TaNAC69-1) in the modulation of root growth in wheat. TaNAC69-1 was predominantly expressed in wheat roots at the early vegetative stage. Overexpression of TaNAC69-1 in wheat roots using OsRSP3 (essentially root-specific) and OsPIP2;3 (root-predominant) promoters resulted in enhanced primary seminal root length and a marked increase in maturity root biomass. Competitive growth analysis under water-limited conditions showed that OsRSP3 promoter-driven TaNAC69-1 transgenic lines produced 32% and 35% more above-ground biomass and grains than wild-type plants, respectively. TaNAC69-1 overexpression in the roots down-regulated the expression of TaSHY2 and TaIAA7, which are from the auxin/IAA (Aux/IAA) transcriptional repressor gene family and are the homologs of negative root growth regulators SHY2/IAA3 and IAA7 in Arabidopsis. The expression of TaSHY2 and TaIAA7 in roots was down-regulated by drought stress and up-regulated by cytokinin treatment, which inhibited root growth. DNA binding and transient expression analyses revealed that TaNAC69-1 bound to the promoters of TaSHY2 and TaIAA7, acted as a transcriptional repressor and repressed the expression of reporter genes driven by the TaSHY2 or TaIAA7 promoter. These data suggest that TaNAC69-1 is a transcriptional repressor of TaSHY2 and TaIAA7 homologous to Arabidopsis negative root growth regulators and is likely to be involved in promoting root elongation in drying soil.

A strong root-specific expression system for stable transgene expression in bread wheat.

KEY MESSAGE: A strong, stable and root-specific expression system was developed from a rice root-specific GLYCINE - RICH PROTEIN 7 promoter for use as an enabling technology for genetic manipulation of wheat root traits. Root systems play an important role in wheat productivity. Genetic manipulation of wheat root traits often requires a root-specific or root-predominant expression system as an essential enabling technology. In this study, we investigated promoters from rice root-specific or root-predominant expressed genes for development of a root expression system in bread wheat. Transient expression analysis using a GREEN FLUORESCENT PROTEIN (GFP) reporter gene driven by rice promoters identified six promoters that were strongly expressed in wheat roots. Extensive organ specificity analysis of three rice promoters in transgenic wheat revealed that the promoter of rice GLYCINE-RICH PROTEIN 7 (OsGRP7) gene conferred a root-specific expression pattern in wheat. Strong GFP fluorescence in the seminal and branch roots of wheat expressing GFP reporter driven by the OsGRP7 promoter was detected in epidermal, cortical and endodermal cells in mature parts of the root. The GFP reporter driven by the promoter of rice METALLOTHIONEIN-LIKE PROTEIN 1 (OsMTL1) gene was mainly expressed in the roots with essentially no expression in the leaf, stem or seed. However, it was also expressed in floral organs including glume, lemma, palea and awn. In contrast, strong expression of rice RCg2 promoter-driven GFP was found in many tissues. The GFP expression driven by these three rice promoters was stable in transgenic wheat plants through three generations (T1-T3) examined. These data suggest that the OsGRP7 promoter can provide a strong, stable and root-specific expression system for use as an enabling technology for genetic manipulation of wheat root traits.

The wheat Lr34 gene provides resistance against multiple fungal pathogens in barley.

The Lr34 gene encodes an ABC transporter and has provided wheat with durable, broad-spectrum resistance against multiple fungal pathogens for over 100 years. Because barley does not have an Lr34 ortholog, we expressed Lr34 in barley to investigate its potential as a broad-spectrum resistance resource in another grass species. We found that introduction of the genomic Lr34 sequence confers resistance against barley leaf rust and barley powdery mildew, two pathogens specific for barley but not virulent on wheat. In addition, the barley lines showed enhanced resistance against wheat stem rust. Transformation with the Lr34 cDNA or the genomic susceptible Lr34 allele did not result in increased resistance. Unlike wheat, where Lr34-conferred resistance is associated with adult plants, the genomic Lr34 transgenic barley lines exhibited multipathogen resistance in seedlings. These transgenic barley lines also developed leaf tip necrosis (LTN) in young seedlings, which correlated with an up-regulation of senescence marker genes and several pathogenesis-related (PR) genes. In wheat, transcriptional expression of Lr34 is highest in adult plants and correlates with increased resistance and LTN affecting the last emerging leaf. The severe phenotype of transgenic Lr34 barley resulted in reduced plant growth and total grain weight. These results demonstrate that Lr34 provides enhanced multipathogen resistance early in barley plant development and implies the conservation of the substrate and mechanism of the LR34 transporter and its molecular action between wheat and barley. With controlled gene expression, the use of Lr34 may be valuable for many cereal breeding programmes, particularly given its proven durability.

Functional variability of the Lr34 durable resistance gene in transgenic wheat.

Breeding for durable disease resistance is challenging, yet essential to improve crops for sustainable agriculture. The wheat Lr34 gene is one of the few cloned, durable resistance genes in plants. It encodes an ATP binding cassette transporter and has been a source of resistance against biotrophic pathogens, such as leaf rust (Puccinina triticina), for over 100 years. As endogenous Lr34 confers quantitative resistance, we wanted to determine the effects of transgenic Lr34 with specific reference to how expression levels affect resistance. Transgenic Lr34 wheat lines were made in two different, susceptible genetic backgrounds. We found that the introduction of the Lr34 resistance allele was sufficient to provide comparable levels of leaf rust resistance as the endogenous Lr34 gene. As with the endogenous gene, we observed resistance in seedlings after cold treatment and in flag leaves of adult plants, as well as Lr34-associated leaf tip necrosis. The transgene-based Lr34 resistance did not involve a hypersensitive response, altered callose deposition or up-regulation of PR genes. Higher expression levels compared to endogenous Lr34 were observed in the transgenic lines both at seedling as well as adult stage and some improvement of resistance was seen in the flag leaf. Interestingly, in one genetic background the transgenic Lr34-based resistance resulted in improved seedling resistance without cold treatment. These data indicate that functional variability in Lr34-based resistance can be created using a transgenic approach.

Overexpression of TaNAC69 leads to enhanced transcript levels of stress up-regulated genes and dehydration tolerance in bread wheat.

NAC proteins are plant-specific transcription factors and enriched with members involved in plant response to drought stress. In this study, we analyzed the expression profiles of TaNAC69 in bread wheat using Affymetrix Wheat Genome Array datasets and quantitative RT-PCR. TaNAC69 expression was positively associated with wheat responses to both abiotic and biotic stresses and was closely correlated with a number of stress up-regulated genes. The functional analyses of TaNAC69 in transgenic wheat showed that TaNAC69 driven by a barley drought-inducible HvDhn4s promoter led to marked drought-inducible overexpression of TaNAC69 in the leaves and roots of transgenic lines. The HvDhn4s:TaNAC69 transgenic lines produced more shoot biomass under combined mild salt stress and water-limitation conditions, had longer root and more root biomass under polyethylene glycol-induced dehydration. Analysis of transgenic lines with constitutive overexpression of TaNAC69 showed the enhanced expression levels of several stress up-regulated genes. DNA-binding assays revealed that TaNAC69 and its rice homolog (ONAC131) were capable of binding to the promoter elements of three rice genes (chitinase, ZIM, and glyoxalase I) and an Arabidopsis glyoxalase I family gene, which are homologs of TaNAC69 up-regulated stress genes. These data suggest that TaNAC69 is involved in regulating stress up-regulated genes and wheat adaptation to drought stress.

Engineering greater aluminium resistance in wheat by over-expressing TaALMT1.

BACKGROUND AND AIMS: Expected increases in world population will continue to make demands on agricultural productivity and food supply. These challenges will only be met by increasing the land under cultivation and by improving the yields obtained on existing farms. Genetic engineering can target key traits to improve crop yields and to increase production on marginal soils. Soil acidity is a major abiotic stress that limits plant production worldwide. The goal of this study was to enhance the acid soil tolerance of wheat by increasing its resistance to Al(3+) toxicity. METHODS: Particle bombardment was used to transform wheat with TaALMT1, the Al(3+) resistance gene from wheat, using the maize ubiquitin promoter to drive expression. TaALMT1 expression, malate efflux and Al(3+) resistance were measured in the T(1) and T(2) lines and compared with the parental line and an Al(3+)-resistant reference genotype, ET8. KEY RESULTS: Nine T(2) lines showed increased TaALMT1 expression, malate efflux and Al(3+) resistance when compared with untransformed controls and null segregant lines. Some T(2) lines displayed greater Al(3+) resistance than ET8 in both hydroponic and soil experiments. CONCLUSIONS: The Al(3+) resistance of wheat was increased by enhancing TaALMT1 expression with biotechnology. This is the first report of a major food crop being stably transformed for greater Al(3+) resistance. Transgenic strategies provide options for increasing food supply on acid soils.

Seed-specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of developing pea cotyledons.

During the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H+ symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H+ symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [(14)C]sucrose influx into cotyledons above wild-type levels was found to be dependent on StSUT1 expression. The transgene significantly increased (approximately 2-fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose influx into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters influence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter fluxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.