ß-Dicarbonyls Facilitate Engineered Microbial Bromoform Biosynthesis.

Ruminant livestock produce around 24% of global anthropogenic methane emissions. Methanogenesis in the animal rumen is significantly inhibited by bromoform, which is abundant in seaweeds of the genus Asparagopsis. This has prompted the development of livestock feed additives based on Asparagopsis to mitigate methane emissions, although this approach alone is unlikely to satisfy global demand. Here we engineer a non-native biosynthesis pathway to produce bromoform in vivo with yeast as an alternative biological source that may enable sustainable, scalable production of bromoform by fermentation. ß-dicarbonyl compounds with low pKa values were identified as essential substrates for bromoform production and enabled bromoform synthesis in engineered Saccharomyces cerevisiae expressing a vanadate-dependent haloperoxidase gene. In addition to providing a potential route to the sustainable biological production of bromoform at scale, this work advances the development of novel microbial biosynthetic pathways for halogenation.

Pooled effector library screening in protoplasts rapidly identifies novel Avr genes.

Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years1-3, the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R-Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) after screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution via genotype surveillance, which in turn will lead to optimized R gene stacking and deployment strategies. This platform should be broadly applicable to many crop pathogens and could potentially be adapted for screening genes involved in other protoplast-selectable traits.

Single amino acid change alters specificity of the multi-allelic wheat stem rust resistance locus SR9.

Most rust resistance genes thus far isolated from wheat have a very limited number of functional alleles. Here, we report the isolation of most of the alleles at wheat stem rust resistance gene locus SR9. The seven previously reported resistance alleles (Sr9a, Sr9b, Sr9d, Sr9e, Sr9f, Sr9g, and Sr9h) are characterised using a synergistic strategy. Loss-of-function mutants and/or transgenic complementation are used to confirm Sr9b, two haplotypes of Sr9e (Sr9e_h1 and Sr9e_h2), Sr9g, and Sr9h. Each allele encodes a highly related nucleotide-binding site leucine-rich repeat (NB-LRR) type immune receptor, containing an unusual long LRR domain, that confers resistance to a unique spectrum of isolates of the wheat stem rust pathogen. The only SR9 protein effective against stem rust pathogen race TTKSK (Ug99), SR9H, differs from SR9B by a single amino acid. SR9B and SR9G resistance proteins are also distinguished by only a single amino acid. The SR9 allelic series found in the B subgenome are orthologs of wheat stem rust resistance gene Sr21 located in the A subgenome with around 85% identity in protein sequences. Together, our results show that functional diversification of allelic variants at the SR9 locus involves single and multiple amino acid changes that recognize isolates of wheat stem rust.

Introgression of the bread wheat D genome encoded Lr34/Yr18/Sr57/Pm38/Ltn1 adult plant resistance gene into Triticum turgidum (durum wheat).

KEY MESSAGE: Lack of function of a D-genome adult plant resistance gene upon introgression into durum wheat. The wheat Lr34/Yr18/Sr57/Pm38/Ltn1 adult plant resistance gene (Lr34), located on chromosome arm 7DS, provides broad spectrum, partial, adult plant resistance to leaf rust, stripe rust, stem rust and powdery mildew. It has been used extensively in hexaploid bread wheat (AABBDD) and conferred durable resistance for many decades. These same diseases also occur on cultivated tetraploid durum wheat and emmer wheat but transfer of D genome sequences to those subspecies is restricted due to very limited intergenomic recombination. Herein we have introgressed the Lr34 gene into chromosome 7A of durum wheat. Durum chromosome substitution line Langdon 7D(7A) was crossed to Cappelli ph1c, a mutant derivative of durum cultivar Cappelli homozygous for a deletion of the chromosome pairing locus Ph1. Screening of BC1F2 plants and their progeny by KASP and PCR markers, 90 K SNP genotyping and cytology identified 7A chromosomes containing small chromosome 7D fragments encoding Lr34. However, in contrast to previous transgenesis experiments in durum wheat, resistance to wheat stripe rust was not observed in either Cappelli/Langdon 7D(7A) or Bansi durum plants carrying this Lr34 encoding segment due to low levels of Lr34 gene expression.

Plant and pathogen genomics: essential approaches for stem rust resistance gene stacks in wheat.

The deployment of disease resistance genes is currently the most economical and environmentally sustainable method of crop protection. However, disease resistance genes can rapidly break down because of constant pathogen evolution, particularly when they are deployed singularly. Polygenic resistance is, therefore, considered the most durable, but combining and maintaining these genes by breeding is a laborious process as effective genes are usually unlinked. The deployment of polygenic resistance with single-locus inheritance is a promising innovation that overcomes these difficulties while enhancing resistance durability. Because of major advances in genomic technologies, increasing numbers of plant resistance genes have been cloned, enabling the development of resistance transgene stacks (RTGSs) that encode multiple genes all located at a single genetic locus. Gene stacks encoding five stem rust resistance genes have now been developed in transgenic wheat and offer both breeding simplicity and potential resistance durability. The development of similar genomic resources in phytopathogens has advanced effector gene isolation and, in some instances, enabled functional validation of individual resistance genes in RTGS. Here, the wheat stem rust pathosystem is used as an illustrative example of how host and pathogen genomic advances have been instrumental in the development of RTGS, which is a strategy applicable to many other agricultural crop species.

A split GAL4 RUBY assay for visual in planta detection of protein-protein interactions.

Protein-protein interactions (PPIs) are a fundamental process in cellular biogenesis. Here we have developed a split GAL4 RUBY assay that enables macroscopically visual PPI detection in plant leaves in real time. Candidate interacting protein partners are fused to specific domains of the yeast GAL4 and herpes simplex virus VP16 transcription factors and transiently expressed in Nicotiana benthamina leaves by Agrobacterium infiltration. PPI, that may be either direct or indirect, results in transcriptional activation of a RUBY reporter gene leading to the production of the highly visual metabolite, betalain, in leaf tissue of living plants. Samples require no processing for in planta visual qualitative assessment, but with very simple processing steps the assay is quantitative. Its accuracy is demonstrated using a series of known interacting protein partners and mutant derivatives including transcription factors, signalling molecules and plant resistance proteins with cognate pathogen effectors. Using this assay, association between the wheat Sr27 stem rust disease resistance protein and corresponding AvrSr27 avirulence effector family produced by the rust pathogen is detected. Interaction is also observed between this resistance protein and the effector encoded by the corresponding avrSr27-3 virulence allele. However, this association appears weaker in the split GAL4 RUBY assay, which coupled with lower avrSr27-3 expression during stem rust infection, likely enables virulent races of the rust pathogen to avoid Sr27-mediated detection.

Haplotype variants of the stripe rust resistance gene Yr28 in Aegilops tauschii.

KEY MESSAGE: Stripe rust resistance gene YrAet672 from Aegilops tauschii accession CPI110672 encodes a nucleotide-binding and leucine-rich repeat domain containing protein similar to YrAS2388 and both these members were haplotypes of Yr28. New sources of host resistance are required to counter the continued emergence of new pathotypes of the wheat stripe rust pathogen Puccinia striiformis Westend. f. sp. tritici Erikss. (Pst). Here, we show that CPI110672, an Aegilops tauschii accession from Turkmenistan, carries a single Pst resistance gene, YrAet672, that is effective against multiple Pst pathotypes, including the four predominant Pst lineages present in Australia. The YRAet672 locus was fine mapped to the short arm of chromosome 4D, and a nucleotide-binding and leucine-rich repeat gene was identified at the locus. A transgene encoding the YrAet672 genomic sequence, but lacking a copy of a duplicated sequence present in the 3' UTR, was transformed into wheat cultivar Fielder and Avocet S. This transgene conferred a weak resistance response, suggesting that the duplicated 3' UTR region was essential for function. Subsequent analyses demonstrated that YrAet672 is the same as two other Pst resistance genes described in Ae. tauschii, namely YrAS2388 and Yr28. They were identified as haplotypes encoding identical protein sequences but are polymorphic in non-translated regions of the gene. Suppression of resistance conferred by YrAet672 and Yr28 in synthetic hexaploid wheat lines (AABBDD) involving Langdon (AABB) as the tetraploid parent was associated with a reduction in transcript accumulation.

Haplotype variants of Sr46 in Aegilops tauschii, the diploid D genome progenitor of wheat.

KEY MESSAGE: Stem rust resistance genes, SrRL5271 and Sr672.1 as well as SrCPI110651, from Aegilops tauschii, the diploid D genome progenitor of wheat, are sequence variants of Sr46 differing by 1-2 nucleotides leading to non-synonymous amino acid substitutions. The Aegilops tauschii (wheat D-genome progenitor) accessions RL 5271 and CPI110672 were identified as resistant to multiple races (including the Ug99) of the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt). This study was conducted to identify the stem rust resistance (Sr) gene(s) in both accessions. Genetic analysis of the resistance in RL 5271 identified a single dominant allele (SrRL5271) controlling resistance, whereas resistance segregated at two loci (SR672.1 and SR672.2) for a cross of CPI110672. Bulked segregant analysis placed SrRL5271 and Sr672.1 in a region on chromosome arm 2DS that encodes Sr46. Molecular marker screening, mapping and genomic sequence analysis demonstrated SrRL5271 and Sr672.1 are alleles of Sr46. The amino acid sequence of SrRL5271 and Sr672.1 is identical but differs from Sr46 (hereafter referred to as Sr46_h1 by following the gene nomenclature in wheat) by a single amino acid (N763K) and is thus designated Sr46_h2. Screening of a panel of Ae. tauschii accessions identified an additional allelic variant that differed from Sr46_h2 by a different amino acid (A648V) and was designated Sr46_h3. By contrast, the protein encoded by the susceptible allele of Ae. tauschii accession AL8/78 differed from these resistance proteins by 54 amino acid substitutions (94% nucleotide sequence gene identity). Cloning and complementation tests of the three resistance haplotypes confirmed their resistance to Pgt race 98-1,2,3,5,6 and partial resistance to Pgt race TTRTF in bread wheat. The three Sr46 haplotypes, with no virulent races detected yet, represent a valuable source for improving stem resistance in wheat.

The barley leaf rust resistance gene Rph3 encodes a predicted membrane protein and is induced upon infection by avirulent pathotypes of Puccinia hordei.

Leaf rust, caused by Puccinia hordei, is an economically significant disease of barley, but only a few major resistance genes to P. hordei (Rph) have been cloned. In this study, gene Rph3 was isolated by positional cloning and confirmed by mutational analysis and transgenic complementation. The Rph3 gene, which originated from wild barley and was first introgressed into cultivated Egyptian germplasm, encodes a unique predicted transmembrane resistance protein that differs from all known plant disease resistance proteins at the amino acid sequence level. Genetic profiles of diverse accessions indicated limited genetic diversity in Rph3 in domesticated germplasm, and higher diversity in wild barley from the Eastern Mediterranean region. The Rph3 gene was expressed only in interactions with Rph3-avirulent P. hordei isolates, a phenomenon also observed for transcription activator-like effector-dependent genes known as executors conferring resistance to Xanthomonas spp. Like known transmembrane executors such as Bs3 and Xa7, heterologous expression of Rph3 in N. benthamiana induced a cell death response. The isolation of Rph3 highlights convergent evolutionary processes in diverse plant-pathogen interaction systems, where similar defence mechanisms evolved independently in monocots and dicots.

Genomics accelerated isolation of a new stem rust avirulence gene-wheat resistance gene pair.

Stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of the global staple crop wheat. Although this disease was largely controlled in the latter half of the twentieth century, new virulent strains of Pgt, such as Ug99, have recently evolved1,2. These strains have caused notable losses worldwide and their continued spread threatens global wheat production. Breeding for disease resistance provides the most cost-effective control of wheat rust diseases3. A number of rust resistance genes have been characterized in wheat and most encode immune receptors of the nucleotide-binding leucine-rich repeat (NLR) class4, which recognize pathogen effector proteins known as avirulence (Avr) proteins5. However, only two Avr genes have been identified in Pgt so far, AvrSr35 and AvrSr50 (refs. 6,7), and none in other cereal rusts8,9. The Sr27 resistance gene was first identified in a wheat line carrying an introgression of the 3R chromosome from Imperial rye10. Although not deployed widely in wheat, Sr27 is widespread in the artificial crop species Triticosecale (triticale), which is a wheat-rye hybrid and is a host for Pgt11,12. Sr27 is effective against Ug99 (ref. 13) and other recent Pgt strains14,15. Here, we identify both the Sr27 gene in wheat and the corresponding AvrSr27 gene in Pgt and show that virulence to Sr27 can arise experimentally and in the field through deletion mutations, copy number variation and expression level polymorphisms at the AvrSr27 locus.

A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes.

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.

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.

A durum wheat adult plant stripe rust resistance QTL and its relationship with the bread wheat Yr80 locus.

KEY MESSAGE: A stripe rust resistance QTL in durum wheat maps near the bread wheat Yr80 locus with the latter reduced to 15 candidate genes. Some wheat adult plant resistance (APR) genes provide partial resistance in the later stages of plant development to rust diseases and are an important component in protecting wheat crops from these fungal pathogens. These genes provide protection in both bread wheat and durum wheat. Here, we have mapped APR to wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici, in a cross between durum cultivars Stewart and Bansi. Two resistance QTLs derived from the Stewart parent were identified in multi-generational field trials. One QTL is located on chromosome 1BL and maps to the previously identified Yr29/Lr46/Sr58/Pm39 multi-pathogen APR locus. The second locus, located on chromosome 3BL, maps near the recently described bread wheat APR gene, Yr80. Fine mapping in durum and bread wheat families shows that the durum 3BL locus and Yr80 are closely located, with the later APR gene reduced to 15 candidate genes present in the Chinese Spring genome sequence. Distorted segregation of the durum 3BL region was observed with the Stewart locus preferentially transmitted through pollen when compared with the equivalent Bansi region.

Plant nonhost resistance: paradigms and new environments.

Nonhost resistance (NHR) protects plants from a large and diverse array of potential phytopathogens. Each phytopathogen can parasitise some plant species, but most plant species are nonhosts that are innately immune due to a series of physical, chemical and inducible defenses these nonadapted pathogens cannot overcome. New evidence supports the NHR paradigm that posits the inability of potential pathogens to colonise nonhost plants is frequently due to molecular incompatibility between pathogen virulence factors and plant cellular targets. While NHR is durable, it is not insurmountable. Environmental changes can facilitate pathogen host jumps or alternatively result in new encounters between previously isolated plant species and pathogens. Climate change is predicted to substantially alter the current distribution of plants and their pathogens which could result in parasitism of new plant species.

The genetic architecture of colonization resistance in Brachypodium distachyon to non-adapted stripe rust (Puccinia striiformis) isolates.

Multilayered defense responses ensure that plants are hosts to only a few adapted pathogens in the environment. The host range of a plant pathogen depends on its ability to fully overcome plant defense barriers, with failure at any single step sufficient to prevent life cycle completion of the pathogen. Puccinia striiformis, the causal agent of stripe rust (=yellow rust), is an agronomically important obligate biotrophic fungal pathogen of wheat and barley. It is generally unable to complete its life cycle on the non-adapted wild grass species Brachypodium distachyon, but natural variation exists for the degree of hyphal colonization by Puccinia striiformis. Using three B. distachyon mapping populations, we identified genetic loci conferring colonization resistance to wheat-adapted and barley-adapted isolates of P. striiformis. We observed a genetic architecture composed of two major effect QTLs (Yrr1 and Yrr3) restricting the colonization of P. striiformis. Isolate specificity was observed for Yrr1, whereas Yrr3 was effective against all tested P. striiformis isolates. Plant immune receptors of the nucleotide binding, leucine-rich repeat (NB-LRR) encoding gene family are present at the Yrr3 locus, whereas genes of this family were not identified at the Yrr1 locus. While it has been proposed that resistance to adapted and non-adapted pathogens are inherently different, the observation of (1) a simple genetic architecture of colonization resistance, (2) isolate specificity of major and minor effect QTLs, and (3) NB-LRR encoding genes at the Yrr3 locus suggest that factors associated with resistance to adapted pathogens are also critical for non-adapted pathogens.

Components of Brachypodium distachyon resistance to nonadapted wheat stripe rust pathogens are simply inherited.

Phytopathogens have a limited range of host plant species that they can successfully parasitise ie. that they are adapted for. Infection of plants by nonadapted pathogens often results in an active resistance response that is relatively poorly characterised because phenotypic variation in this response often does not exist within a plant species, or is too subtle for genetic dissection. In addition, complex polygenic inheritance often underlies these resistance phenotypes and mutagenesis often does not impact upon this resistance, presumably due to genetic or mechanistic redundancy. Here it is demonstrated that phenotypic differences in the resistance response of Brachypodium distachyon to the nonadapted wheat stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst) are genetically tractable and simply inherited. Two dominant loci were identified on B. distachyon chromosome 4 that each reduce attempted Pst colonisation compared with sib and parent lines without these loci. One locus (Yrr1) is effective against diverse Australian Pst isolates and present in two B. distachyon mapping families as a conserved region that was reduced to 5 candidate genes by fine mapping. A second locus, Yrr2, shows Pst race-specificity and encodes a disease resistance gene family typically associated with host plant resistance. These data indicate that some components of resistance to nonadapted pathogens are genetically tractable in some instances and may mechanistically overlap with host plant resistance to avirulent adapted pathogens.

Modulator material impact on chirp, DSP, and performance in coherent digital links: comparison of the lithium niobate, indium phosphide, and silicon platforms.

We characterize the impact of the modulator material on chirp, digital signal processing (DSP) algorithms and system-level performance in coherent digital optical links. We compare theoretically, in simulations and experimentally the lithium niobate (LiNbO3), indium phosphide (InP) and silicon (Si) integrated platforms. Distortions to vector diagrams are traced back to modulation physics, and are interpreted as quadrature crosstalk. In a back-to-back BPSK setup with an RF drive signal amplitude of 1.5Vπ, we measure chirp parameters α of ~0, 0.10 and 0.06 and error vector magnitude EVMRMS of 5.3%, 9.4% and 10.6% with the LiNbO3, InP and Si modulators respectively. Both α and EVMRMS are found to scale with the RF signal amplitude. In simulations, using a polynomial fit over a sinusoidal fit when pre-compensating the Si modulator transfer function slightly improves EVM (-0.6%). We also show that Si-related distortions can impact the efficiency of symbol timing recovery. In conclusion, phase and attenuation distortions in InP and Si modulators deteriorate the overall performance in coherent links, and cannot be neglected for large RF signal amplitudes. These results will benefit the optical communications community.