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.     

The wheat durable,multipathogen resistance gene Lr34 confers partial blast resistance in rice

The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain‐of‐function mutations in an ATP‐binding cassette transporter gene. An Lr34‐like fungal disease resistance with a similar broad‐spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34‐expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence‐based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad‐spectrum disease resistance against the most devastating fungal disease of rice.     

Pathogen‐inducible Ta‐Lr34res expression in heterologous barley confers disease resistance without negative pleiotropic effects

Plant diseases are a serious threat to crop production. The informed use of naturally occurring disease resistance in plant breeding can greatly contribute to sustainably reduce yield losses caused by plant pathogens. The Ta‐Lr34res gene encodes an ABC transporter protein and confers partial, durable, and broad spectrum resistance against several fungal pathogens in wheat. Transgenic barley lines expressing Ta‐Lr34res showed enhanced resistance against powdery mildew and leaf rust of barley. While Ta‐Lr34res is only active at adult stage in wheat, Ta‐Lr34res was found to be highly expressed already at the seedling stage in transgenic barley resulting in severe negative effects on growth. Here, we expressed Ta‐Lr34res under the control of the pathogen‐inducible Hv‐Ger4c promoter in barley. Sixteen independent barley transformants showed strong resistance against leaf rust and powdery mildew. Infection assays and growth parameter measurements were performed under standard glasshouse and near‐field conditions using a convertible glasshouse. Two Hv‐Ger4c::Ta‐Lr34res transgenic events were analysed in detail. Plants of one transformation event had similar grain production compared to wild‐type under glasshouse and near‐field conditions. Our results showed that negative effects caused by constitutive high expression of Ta‐Lr34res driven by the endogenous wheat promoter in barley can be eliminated by inducible expression without compromising disease resistance. These data demonstrate that Ta‐Lr34res is agronomically useful in barley. We conclude that the generation of a large number of transformants in different barley cultivars followed by early field testing will allow identifying barley lines suitable for breeding.     

The wheat Lr34 multipathogen resistance gene confers resistance to anthracnose and rust in sorghum

The ability of the wheat Lr34 multipathogen resistance gene (Lr34res) to function across a wide taxonomic boundary was investigated in transgenic Sorghum bicolor. Increased resistance to sorghum rust and anthracnose disease symptoms following infection with the biotrophic pathogen Puccinia purpurea and the hemibiotroph Colletotrichum sublineolum, respectively, occurred in transgenic plants expressing the Lr34res ABC transporter. Transgenic sorghum lines that highly expressed the wheat Lr34res gene exhibited immunity to sorghum rust compared to the low‐expressing single copy Lr34res genotype that conferred partial resistance. Pathogen‐induced pigmentation mediated by flavonoid phytoalexins was evident on transgenic sorghum leaves following P. purpurea infection within 24–72 h, which paralleled Lr34res gene expression. Elevated expression of flavone synthase II, flavanone 4‐reductase and dihydroflavonol reductase genes which control the biosynthesis of flavonoid phytoalexins characterized the highly expressing Lr34res transgenic lines 24‐h post‐inoculation with P. purpurea. Metabolite analysis of mesocotyls infected with C. sublineolum showed increased levels of 3‐deoxyanthocyanidin metabolites were associated with Lr34res expression, concomitant with reduced symptoms of anthracnose.     

The Lr34 adult plant rust resistance gene provides seedling resistance in durum wheat without senescence

The hexaploid wheat (Triticum aestivum) adult plant resistance gene, Lr34/Yr18/Sr57/Pm38/Ltn1, provides broad‐spectrum resistance to wheat leaf rust (Lr34), stripe rust (Yr18), stem rust (Sr57) and powdery mildew (Pm38) pathogens, and has remained effective in wheat crops for many decades. The partial resistance provided by this gene is only apparent in adult plants and not effective in field‐grown seedlings. Lr34 also causes leaf tip necrosis (Ltn1) in mature adult plant leaves when grown under field conditions. This D genome‐encoded bread wheat gene was transferred to tetraploid durum wheat (T. turgidum) cultivar Stewart by transformation. Transgenic durum lines were produced with elevated gene expression levels when compared with the endogenous hexaploid gene. Unlike nontransgenic hexaploid and durum control lines, these transgenic plants showed robust seedling resistance to pathogens causing wheat leaf rust, stripe rust and powdery mildew disease. The effectiveness of seedling resistance against each pathogen correlated with the level of transgene expression. No evidence of accelerated leaf necrosis or up‐regulation of senescence gene markers was apparent in these seedlings, suggesting senescence is not required for Lr34 resistance, although leaf tip necrosis occurred in mature plant flag leaves. Several abiotic stress‐response genes were up‐regulated in these seedlings in the absence of rust infection as previously observed in adult plant flag leaves of hexaploid wheat. Increasing day length significantly increased Lr34 seedling resistance. These data demonstrate that expression of a highly durable, broad‐spectrum adult plant resistance gene can be modified to provide seedling resistance in durum wheat.     

The durable wheat disease resistance gene Lr34 confers common rust and northern corn leaf blight resistance in maize

Maize (corn) is one of the most widely grown cereal crops globally. Fungal diseases of maize cause significant economic damage by reducing maize yields and by increasing input costs for disease management. The most sustainable control of maize diseases is through the release and planting of maize cultivars with durable disease resistance. The wheat gene Lr34 provides durable and partial field resistance against multiple fungal diseases of wheat, including three wheat rust pathogens and wheat powdery mildew. Because of its unique qualities, Lr34 became a cornerstone in many wheat disease resistance programmes. The Lr34 resistance is encoded by a rare variant of an ATP‐binding cassette (ABC) transporter that evolved after wheat domestication. An Lr34‐like disease resistance phenotype has not been reported in other cereal species, including maize. Here, we transformed the Lr34 resistance gene into the maize hybrid Hi‐II. Lr34‐expressing maize plants showed increased resistance against the biotrophic fungal disease common rust and the hemi‐biotrophic disease northern corn leaf blight. Furthermore, the Lr34‐expressing maize plants developed a late leaf tip necrosis phenotype, without negative impact on plant growth. With this and previous reports, it could be shown that Lr34 is effective against various biotrophic and hemi‐biotrophic diseases that collectively parasitize all major cereal crop species.     

Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

Lr34 multi-pathogen resistance ABC transporter: molecular analysis of homoeologous and orthologous genes in hexaploid wheat and other grass species

The Triticum aestivum (bread wheat) disease resistance gene Lr34 confers durable, race non-specific protection against three fungal pathogens, and has been a highly relevant gene for wheat breeding since the green revolution. Lr34, located on chromosome 7D, encodes an ATP-binding cassette (ABC) transporter. Both wheat cultivars with and without Lr34-based resistance encode a putatively functional protein that differ by only two amino acid polymorphisms. In this study, we focused on the identification and characterization of homoeologous and orthologous Lr34 genes in hexaploid wheat and other grasses. In hexaploid wheat we found an expressed and putatively functional Lr34 homoeolog located on chromosome 4A, designated Lr34-B. Another homoeologous Lr34 copy, located on chromosome 7A, was disrupted by the insertion of repetitive elements. Protein sequences of LR34-B and LR34 were 97% identical. Orthologous Lr34 genes were detected in the genomes of Oryza sativa (rice) and Sorghum bicolor (sorghum). Zea mays (maize), Brachypodium distachyon and Hordeum vulgare (barley) lacked Lr34 orthologs, indicating independent deletion of this particular ABC transporter. Lr34 was part of a gene-rich island on the wheat D genome. We found gene colinearity on the homoeologous A and B genomes of hexaploid wheat, but little microcolinearity in other grasses. The homoeologous LR34-B protein and the orthologs from rice and sorghum have the susceptible haplotype for the two critical polymorphisms distinguishing the LR34 proteins from susceptible and resistant wheat cultivars. We conclude that the particular Lr34-haplotype found in resistant wheat cultivars is unique. It probably resulted from functional gene diversification that occurred after the polyploidization event that was at the origin of cultivated bread wheat.     

Identification of a STS marker linked to the Aegilops speltoides-derived leaf rust resistance gene Lr28 in wheat

 A sequence-tagged-site (STS) marker is reported linked to Lr28, a leaf rust resistance gene in wheat. RAPD (random amplified polymorphic DNA) analysis of near-isogenic lines (NILs) of Lr28 in eight varietal backgrounds was carried out using random primers. Genomic DNA enriched for low-copy sequences was used for RAPD analysis to overcome the lack of reproducibility due to the highly repetitive DNA sequences present in wheat. Of 80 random primers tested on the enriched DNA, one RAPD marker distinguished the NILs and the donor parent from the susceptible recurrent parents. The additional band present in resistant lines was cloned, sequenced, and STS primers specific for Lr28 were designed. The STS marker (Indian patent pending: 380 Del98) was further confirmed by bulk segregation analysis of F₃ families. It was consistently present in the NILs, the resistant F₃ bulk and the resistant F₃ lines, but was absent in recurrent parents, the susceptible F₃ bulk and the susceptible F₃ lines. Received: 20 February 1998 / Accepted: 4 March 1998     

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol‐3‐phosphate‐binding proteins

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol‐3‐phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P‐binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P‐binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P‐binding site, or by a secreted PI4P‐binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P‐binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P‐binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.     

The evolution of resistance against good and bad infections

Opportunities for genetic exchange are abundant between bacteria and foreign genetic elements (FGEs) such as conjugative plasmids, transposable elements and bacteriophages. The genetic novelty that may arise from these forms of genetic exchange is potentially beneficial to bacterial hosts, but there are also potential costs, which may be considerable in the case of phage infection. Some bacterial resistance mechanisms target both beneficial and deleterious forms of genetic exchange. Using a general epidemiological model, we explored under which conditions such resistance mechanisms may evolve. We considered a population of hosts that may be infected by FGEs that either confer a benefit or are deleterious to host fitness, and we analysed the epidemiological and evolutionary outcomes of resistance evolving under different cost/benefit scenarios. We show that the degree of co‐infection between these two types of infection is particularly important in determining the evolutionarily stable level of host resistance. We explore these results using the example of CRISPR‐Cas, a form of bacterial immunity that targets a variety of FGEs, and we show the potential role of bacteriophage infection in selecting for resistance mechanisms that in turn limit the acquisition of plasmid‐borne antibiotic resistance. Finally, beyond microbes, we discuss how endosymbiotic associations may have shaped the evolution of host immune responses to pathogens.     

Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene

Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat‐growing areas in China. Because it is vectored by the fungus‐like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co‐transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12‐1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12‐1 showed broad‐spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild‐type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus‐derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad‐spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.     

The genetic architecture of defence as resistance to and tolerance of bacterial infection in Drosophila melanogaster

Defence against pathogenic infection can take two forms: resistance and tolerance. Resistance is the ability of the host to limit a pathogen burden, whereas tolerance is the ability to limit the negative consequences of infection at a given level of infection intensity. Evolutionarily, a tolerance strategy that is independent of resistance could allow the host to avoid mounting a costly immune response and, theoretically, to avoid a co‐evolutionary arms race between pathogen virulence and host resistance. Biomedically, understanding the mechanisms of tolerance and how they relate to resistance could potentially yield treatment strategies that focus on health improvement instead of pathogen elimination. To understand the impact of tolerance on host defence and identify genetic variants that determine host tolerance, we defined genetic variation in tolerance as the residual deviation from a binomial regression of fitness under infection against infection intensity. We then performed a genomewide association study to map the genetic basis of variation in resistance to and tolerance of infection by the bacterium Providencia rettgeri. We found a positive genetic correlation between resistance and tolerance, and we demonstrated that the level of resistance is highly predictive of tolerance. We identified 30 loci that predict tolerance, many of which are in genes involved in the regulation of immunity and metabolism. We used RNAi to confirm that a subset of mapped genes have a role in defence, including putative wound repair genes grainy head and debris buster. Our results indicate that tolerance is not an independent strategy from resistance, but that defence arises from a collection of physiological processes intertwined with canonical immunity and resistance.     

CRISPR/SpCas9-mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity,transcriptional gene silencing and plant growth

The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9-mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium-mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9-induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo-/heteromers with other HvMORCs and interact with components of the RNA-directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.     

Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad‐spectrum resistance against insects,pathogens and abiotic stresses

Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid‐expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.     

Variation in resistance to multiple pathogen species: anther smuts of Silene uniflora

The occurrence of multiple pathogen species on a shared host species is unexpected when they exploit the same micro‐niche within the host individual. One explanation for such observations is the presence of pathogen‐specific resistances segregating within the host population into sites that are differentially occupied by the competing pathogens. This study used experimental inoculations to test whether specific resistances may contribute to the maintenance of two species of anther‐smut fungi, Microbotryum silenes‐inflatae and Microbotryum lagerheimii, in natural populations of Silene uniflora in England and Wales. Overall, resistance to the two pathogens was strongly positively correlated among host populations and to a lesser degree among host families within populations. A few instances of specific resistance were also observed and confirmed by replicated inoculations. The results suggest that selection for resistance to one pathogen may protect the host from the emergence via host shifts of related pathogen species, and conversely that co‐occurrence of two species of pathogens may be dependent on the presence of host genotypes susceptible to both.