The complex relationship between disease resistance and yield in crops.

In plants, growth and defence are controlled by many molecular pathways that are antagonistic to one another. This results in a 'growth-defence trade-off', where plants temporarily reduce growth in response to pests or diseases. Due to this antagonism, genetic variants that improve resistance often reduce growth and vice versa. Therefore, in natural populations, the most disease resistant individuals are often the slowest growing. In crops, slow growth may translate into a yield penalty, but resistance is essential for protecting yield in the presence of disease. Therefore, plant breeders must balance these traits to ensure optimal yield potential and yield stability. In crops, both qualitative and quantitative disease resistance are often linked with genetic variants that cause yield penalties, but this is not always the case. Furthermore, both crop yield and disease resistance are complex traits influenced by many aspects of the plant's physiology, morphology and environment, and the relationship between the molecular growth-defence trade-off and disease resistance-yield antagonism is not well-understood. In this article, we highlight research from the last 2 years on the molecular mechanistic basis of the antagonism between defence and growth. We then discuss the interaction between disease resistance and crop yield from a breeding perspective, outlining the complexity and nuances of this relationship and where research can aid practical methods for simultaneous improvement of yield potential and disease resistance.

Inter-organellar nucleic acid communication by a mitochondrial tRNA regulates nuclear metabolic transcription.

Efficient communication between mitochondria and the nucleus underlies homoeostatic metabolic control, though the involved mitochondrial factors and their mechanisms are poorly defined. Here, we report the surprising detection of multiple mitochondrial-derived transfer RNAs (mito-tRNAs) within the nuclei of human cells. Focused studies of nuclear-transported mito-tRNA-asparagine (mtAsn) revealed that its cognate charging enzyme (NARS2) is also present in the nucleus. MtAsn promoted interaction of NARS2 with histone deacetylase 2 (HDAC2), and repressed HDAC2 association with specific chromatin loci. Perturbation of this axis using antisense oligonucleotides promoted nucleotide biogenesis and enhanced breast cancer growth, and RNA and nascent transcript sequencing demonstrated specific alterations in the transcription of nuclear genes. These findings uncover nucleic-acid mediated communication between two organelles and the existence of a machinery for nuclear gene regulation by a mito-tRNA that restricts tumor growth through metabolic control. Highlights: Multiple mitochondrial-derived tRNAs are detected in human cell nucleiMtAsn promotes binding between NARS2 and HDAC2Metabolic alterations driven by mtAsn impact cell proliferationMtAsn inhibition releases HDAC2 to bind and transcriptionally regulate multiple nuclear genes.

Genome-wide identification of Sclerotinia sclerotiorum small RNAs and their endogenous targets.

BACKGROUND: Several phytopathogens produce small non-coding RNAs of approximately 18-30 nucleotides (nt) which post-transcriptionally regulate gene expression. Commonly called small RNAs (sRNAs), these small molecules were also reported to be present in the necrotrophic pathogen Sclerotinia sclerotiorum. S. sclerotiorum causes diseases in more than 400 plant species, including the important oilseed crop Brassica napus. sRNAs can further be classified as microRNAs (miRNAs) and short interfering RNAs (siRNAs). Certain miRNAs can activate loci that produce further sRNAs; these secondary sRNA-producing loci are called 'phased siRNA' (PHAS) loci and have only been described in plants. To date, very few studies have characterized sRNAs and their endogenous targets in S. sclerotiorum. RESULTS: We used Illumina sequencing to characterize sRNAs from fungal mycelial mats of S. sclerotiorum spread over B. napus leaves. In total, eight sRNA libraries were prepared from in vitro, 12 h post-inoculation (HPI), and 24 HPI mycelial mat samples. Cluster analysis identified 354 abundant sRNA clusters with reads of more than 100 Reads Per Million (RPM). Differential expression analysis revealed upregulation of 34 and 57 loci at 12 and 24 HPI, respectively, in comparison to in vitro samples. Among these, 25 loci were commonly upregulated. Altogether, 343 endogenous targets were identified from the major RNAs of 25 loci. Almost 88% of these targets were annotated as repeat element genes, while the remaining targets were non-repeat element genes. Fungal degradome reads confirmed cleavage of two transposable elements by one upregulated sRNA. Altogether, 24 milRNA loci were predicted with both mature and milRNA* (star) sequences; these are both criteria associated previously with experimentally verified miRNAs. Degradome sequencing data confirmed the cleavage of 14 targets. These targets were related to repeat element genes, phosphate acetyltransferases, RNA-binding factor, and exchange factor. A PHAS gene prediction tool identified 26 possible phased interfering loci with 147 phasiRNAs from the S. sclerotiorum genome, suggesting this pathogen might produce sRNAs that function similarly to miRNAs in higher eukaryotes. CONCLUSIONS: Our results provide new insights into sRNA populations and add a new resource for the study of sRNAs in S. sclerotiorum.

Till death do us pair: Co-evolution of plant-necrotroph interactions.

Plants use programmed cell death as a potent defense response against biotrophic pathogens that require living host cells to thrive. However, cell death can promote infection by necrotrophic pathogens. This discrepancy creates specific co-evolutionary dynamics in the interaction between plants and necrotrophs. Necrotrophic pathogens produce diverse cell death-inducing effectors that act redundantly on several plant targets and sometimes suppress plant immune responses as an additional function. Plants use surface receptors that recognize necrotrophic effectors to increase quantitative disease resistance, some of which evolved independently in several plant lineages. Co-evolution has shaped molecular mechanisms involved in plant-necrotroph interactions into robust systems, relying on degenerate and multifunctional modules, general-purpose components, and compartmentalized functioning.

The pursuit of genetic gain in agricultural crops through the application of machine-learning to genomic prediction.

Current practice in agriculture applies genomic prediction to assist crop breeding in the analysis of genetic marker data. Genomic selection methods typically use linear mixed models, but using machine-learning may provide further potential for improved selection accuracy, or may provide additional information. Here we describe SelectML, an automated pipeline for testing and comparing the performance of a range of linear mixed model and machine-learning-based genomic selection methods. We demonstrate the use of SelectML on an in silico-generated marker dataset which simulated a randomly-sampled (mixed) and an unevenly-sampled (unbalanced) population, comparing the relative performance of various methods included in SelectML on the two datasets. Although machine-learning based methods performed similarly overall to linear mixed models, they performed worse on the mixed dataset and marginally better on the unbalanced dataset, being more affected than linear mixed models by the imposed sampling bias. SelectML can assist in the training, comparison, and selection of genomic selection models, and is available from https://github.com/darcyabjones/selectml.

Surface frustration re-patterning underlies the structural landscape and evolvability of fungal orphan candidate effectors.

Pathogens secrete effector proteins to subvert host physiology and cause disease. Effectors are engaged in a molecular arms race with the host resulting in conflicting evolutionary constraints to manipulate host cells without triggering immune responses. The molecular mechanisms allowing effectors to be at the same time robust and evolvable remain largely enigmatic. Here, we show that 62 conserved structure-related families encompass the majority of fungal orphan effector candidates in the Pezizomycotina subphylum. These effectors diversified through changes in patterns of thermodynamic frustration at surface residues. The underlying mutations tended to increase the robustness of the overall effector protein structure while switching potential binding interfaces. This mechanism could explain how conserved effector families maintained biological activity over long evolutionary timespans in different host environments and provides a model for the emergence of sequence-unrelated effector families with conserved structures.

Multiple Dermatomyofibromas in a Patient with Ehlers-Danlos Syndrome.

Dermatomyofibromas are a rare, benign, acquired neoplasm with less than 150 reported cases worldwide. The etiologic factors that contribute to the development of these lesions are currently unknown. To our knowledge, there have been only six previously reported cases of patients presenting with multiple dermatomyofibromas, and in each of these cases, there were less than ten lesions present. Herein, we describe a patient who developed more than 100 dermatomyofibromas over a period of years, and we argue that the patient's concurrent Ehlers-Danlos syndrome could have contributed to this unique presentation by inducing an increased fibroblast-to-myofibroblast transition.

Diversity of fatty acid biosynthesis genes across the soybean pangenome.

Soybean (Glycine max) is a major crop that contributes more than half of global oilseed production. Much research has been directed towards improvement of the fatty acid profile of soybean seeds through marker assisted breeding. Recently published soybean pangenomes, based on thousands of soybean lines, provide an opportunity to identify new alleles that may be involved in fatty acid biosynthesis. In this study, we identify fatty acid biosynthesis genes in soybean pangenomes based on sequence identity with known genes and examine their sequence diversity across diverse soybean collections. We find three possible instances of a gene missing in wild soybean, including FAD8 and FAD2-2D, which may be involved in oleic and linoleic acid desaturation, respectively, although we recommend follow-up research to verify the absence of these genes. More than half of the 53 fatty acid biosynthesis genes identified contained missense variants, including one linked with a previously identified QTL for oil quality. These variants were present in multiple studies based on either short read mappings or alignment of reference grade genomes. Missense variants were found in previously characterized genes including FAD2-1A and FAD2-1B, both of which are involved in desaturation of oleic acid, as well as uncharacterized candidate fatty acid biosynthesis genes. We find that the frequency of missense alleles in fatty acid biosynthesis genes has been reduced significantly more than the global average frequency of missense mutations during domestication, and missense variation in some genes is near absent in modern cultivars. This could be due to the selection for fatty acid profiles in seed, though future work should be conducted towards understanding the phenotypic impacts of these variants.

Loss of Tbx3 in Mouse Eye Causes Retinal Angiogenesis Defects Reminiscent of Human Disease.

Purpose: Familial exudative vitreoretinopathy (FEVR) and Norrie disease are examples of genetic disorders in which the retinal vasculature fails to fully form (hypovascular), leading to congenital blindness. While studying the role of a factor expressed during retinal development, T-box factor Tbx3, we discovered that optic cup loss of Tbx3 caused the retina to become hypovascular. The purpose of this study was to characterize how loss of Tbx3 affects retinal vasculature formation. Methods: Conditional removal of Tbx3 from both retinal progenitors and astrocytes was done using the optic cup-Cre recombinase driver BAC-Dkk3-Cre and was analyzed using standard immunohistochemical techniques. Results: With Tbx3 loss, the retinas were hypovascular, as seen in patients with retinopathy of prematurity (ROP) and FEVR. Retinal vasculature failed to form the stereotypic tri-layered plexus in the dorsal-temporal region. Astrocyte precursors were reduced in number and failed to form a lattice at the dorsal-temporal edge. We next examined retinal ganglion cells, as they have been shown to play a critical role in retinal angiogenesis. We found that melanopsin expression and Islet1/2-positive retinal ganglion cells were reduced in the dorsal half of the retina. In previous studies, the loss of melanopsin has been linked to hyaloid vessel persistence, which we also observed in the Tbx3 conditional knockout (cKO) retinas, as well as in infants with ROP or FEVR. Conclusions: To the best of our knowledge, these studies are the first demonstration that Tbx3 is required for normal mammalian eye formation. Together, the results provide a potential genetic model for retinal hypovascular diseases.

The broad host range pathogen Sclerotinia sclerotiorum produces multiple effector proteins that induce host cell death intracellularly.

Sclerotinia sclerotiorum is a broad host range necrotrophic fungal pathogen, which causes disease on many economically important crop species. S. sclerotiorum has been shown to secrete small effector proteins to kill host cells and acquire nutrients. We set out to discover novel necrosis-inducing effectors and characterize their activity using transient expression in Nicotiana benthamiana leaves. Five intracellular necrosis-inducing effectors were identified with differing host subcellular localization patterns, which were named intracellular necrosis-inducing effector 1-5 (SsINE1-5). We show for the first time a broad host range pathogen effector, SsINE1, that uses an RxLR-like motif to enter host cells. Furthermore, we provide preliminary evidence that SsINE5 induces necrosis via an NLR protein. All five of the identified effectors are highly conserved in globally sourced S. sclerotiorum isolates. Taken together, these results advance our understanding of the virulence mechanisms employed by S. sclerotiorum and reveal potential avenues for enhancing genetic resistance to this damaging fungal pathogen.

Association Mapping Combined with Whole Genome Sequencing Data Reveals Candidate Causal Variants for Sclerotinia Stem Rot Resistance in Brassica napus.

Canola (Brassica napus) yield can be significantly reduced by the disease sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum, a necrotrophic fungal pathogen with an unusually large host range. Breeding cultivars that are physiologically resistant to SSR is desirable to enhance crop productivity. However, the development of resistant varieties has proved challenging due to the highly polygenic nature of S. sclerotiorum resistance. Here, we identified regions of the B. napus genome associated with SSR resistance using data from a previous study by association mapping. We then validated their contribution to resistance in a follow-up screen. This follow-up screen also confirmed high levels of SSR resistance in several genotypes from the previous study. Using publicly available whole genome sequencing data for a panel of 83 B. napus genotypes, we identified nonsynonymous polymorphisms linked to the SSR resistance loci. A qPCR analysis showed that two of the genes containing these polymorphisms were transcriptionally responsive to S. sclerotiorum infection. In addition, we provide evidence that homologues of three of the candidate genes contribute to resistance in the model Brassicaceae species Arabidopsis thaliana. The identification of resistant germplasm and candidate genomic loci associated with resistance are important findings that can be exploited by breeders to improve the genetic resistance of canola varieties.

Small RNA Analyses of a Ceratobasidium Isolate Infected with Three Endornaviruses.

Isolates of three endornavirus species were identified co-infecting an unidentified species of Ceratobasidium, itself identified as a symbiont from within the roots of a wild plant of the terrestrial orchid Pterostylis vittata in Western Australia. Isogenic lines of the fungal isolate lacking all three mycoviruses were derived from the virus-infected isolate. To observe how presence of endornaviruses influenced gene expression in the fungal host, we sequenced fungus-derived small RNA species from the virus-infected and virus-free isogenic lines and compared them. The presence of mycoviruses influenced expression of small RNAs. Of the 3272 fungus-derived small RNA species identified, the expression of 9.1% (300 of 3272) of them were up-regulated, and 0.6% (18 of 3272) were down-regulated in the presence of the viruses. Fourteen novel micro-RNA-like RNAs (Cer-milRNAs) were predicted. Gene target prediction of the differentially expressed Cer-milRNAs was quite ambiguous; however, fungal genes involved in transcriptional regulation, catalysis, molecular binding, and metabolic activities such as gene expression, DNA metabolic processes and regulation activities were differentially expressed in the presence of the mycoviruses.

The evolutionary and molecular features of the broad-host-range plant pathogen Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of plant species, including many of the world's most important crops. Key features of S. sclerotiorum include its extraordinary host range, preference for dicotyledonous plants, relatively slow evolution, and production of protein effectors that are active in multiple host species. Plant resistance to this pathogen is highly complex, typically involving numerous polymorphisms with infinitesimally small effects, which makes resistance breeding a major challenge. Due to its economic significance, S. sclerotiorum has been subjected to a large amount of molecular and evolutionary research. In this updated pathogen profile, we review the evolutionary and molecular features of S. sclerotiorum and discuss avenues for future research into this important species.

Identification of Sclerotinia stem rot resistance quantitative trait loci in a chickpea (Cicer arietinum) recombinant inbred line population.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum , is one of the most economically devastating diseases in chickpea (Cicer arietinum L.). No complete resistance is available in chickpea to this disease, and the inheritance of partial resistance is not understood. Two hundred F7 recombinant inbred lines (RILs) derived from a cross between a partially resistant variety PBA HatTrick, and a highly susceptible variety Kyabra were characterised for their responses to SSR inoculation. Quantitative trait locus (QTL) analysis was conducted for the area under the disease progress curve (AUDPC) after RIL infection with S. sclerotiorum . Four QTLs on chromosomes, Ca4 (qSSR4-1, qSSR4-2), Ca6 (qSSR6-1) and Ca7 (qSSR7-1), individually accounted for between 4.2 and 15.8% of the total estimated phenotypic variation for the response to SSR inoculation. Candidate genes located in these QTL regions are predicted to be involved in a wide range of processes, including phenylpropanoid biosynthesis, plant-pathogen interaction, and plant hormone signal transduction. This is the first study investigating the inheritance of resistance to S. sclerotiorum in chickpea. Markers associated with the identified QTLs could be employed for marker-assisted selection in chickpea breeding.

fIdentification of B. napus small RNAs responsive to infection by a necrotrophic pathogen.

BACKGROUND: Small RNAs are short non-coding RNAs that are key gene regulators controlling various biological processes in eukaryotes. Plants may regulate discrete sets of sRNAs in response to pathogen attack. Sclerotinia sclerotiorum is an economically important pathogen affecting hundreds of plant species, including the economically important oilseed B. napus. However, there are limited studies on how regulation of sRNAs occurs in the S. sclerotiorum and B. napus pathosystem. RESULTS: We identified different classes of sRNAs from B. napus using high throughput sequencing of replicated mock and infected samples at 24 h post-inoculation (HPI). Overall, 3999 sRNA loci were highly expressed, of which 730 were significantly upregulated during infection. These 730 up-regulated sRNAs targeted 64 genes, including disease resistance proteins and transcriptional regulators. A total of 73 conserved miRNA families were identified in our dataset. Degradome sequencing identified 2124 cleaved mRNA products from these miRNAs from combined mock and infected samples. Among these, 50 genes were specific to infection. Altogether, 20 conserved miRNAs were differentially expressed and 8 transcripts were cleaved by the differentially expressed miRNAs miR159, miR5139, and miR390, suggesting they may have a role in the S. sclerotiorum response. A miR1885-triggered disease resistance gene-derived secondary sRNA locus was also identified and verified with degradome sequencing. We also found further evidence for silencing of a plant immunity related ethylene response factor gene by a novel sRNA using 5'-RACE and RT-qPCR. CONCLUSIONS: The findings in this study expand the framework for understanding the molecular mechanisms of the S. sclerotiorum and B. napus pathosystem at the sRNA level.

Positive Selection of Transcription Factors Is a Prominent Feature of the Evolution of a Plant Pathogenic Genus Originating in the Miocene.

Tests based on the dN/dS statistic are used to identify positive selection of nonsynonymous polymorphisms. Using these tests on alignments of all orthologs from related species can provide insights into which gene categories have been most frequently positively selected. However, longer alignments have more power to detect positive selection, creating a detection bias that could create misleading results from functional enrichment tests. Most studies of positive selection in plant pathogens focus on genes with specific virulence functions, with little emphasis on broader molecular processes. Furthermore, no studies in plant pathogens have accounted for detection bias due to alignment length when performing functional enrichment tests. To address these research gaps, we analyze 12 genomes of the phytopathogenic fungal genus Botrytis, including two sequenced in this study. To establish a temporal context, we estimated fossil-calibrated divergence times for the genus. We find that Botrytis likely originated 16-18 Ma in the Miocene and underwent continuous radiation ending in the Pliocene. An untargeted scan of Botrytis single-copy orthologs for positive selection with three different statistical tests uncovered evidence for positive selection among proteases, signaling proteins, CAZymes, and secreted proteins. There was also a strong overrepresentation of transcription factors among positively selected genes. This overrepresentation was still apparent after two complementary controls for detection bias due to sequence length. Positively selected sites were depleted within DNA-binding domains, suggesting changes in transcriptional responses to internal and external cues or protein-protein interactions have undergone positive selection more frequently than changes in promoter fidelity.

Analysis of differentially expressed Sclerotinia sclerotiorum genes during the interaction with moderately resistant and highly susceptible chickpea lines.

BACKGROUND: Sclerotinia sclerotiorum, the cause of Sclerotinia stem rot (SSR), is a host generalist necrotrophic fungus that can cause major yield losses in chickpea (Cicer arietinum) production. This study used RNA sequencing to conduct a time course transcriptional analysis of S. sclerotiorum gene expression during chickpea infection. It explores pathogenicity and developmental factors employed by S. sclerotiorum during interaction with chickpea. RESULTS: During infection of moderately resistant (PBA HatTrick) and highly susceptible chickpea (Kyabra) lines, 9491 and 10,487 S. sclerotiorum genes, respectively, were significantly differentially expressed relative to in vitro. Analysis of the upregulated genes revealed enrichment of Gene Ontology biological processes, such as oxidation-reduction process, metabolic process, carbohydrate metabolic process, response to stimulus, and signal transduction. Several gene functional categories were upregulated in planta, including carbohydrate-active enzymes, secondary metabolite biosynthesis clusters, transcription factors and candidate secreted effectors. Differences in expression of four S. sclerotiorum genes on varieties with different levels of susceptibility were also observed. CONCLUSION: These findings provide a framework for a better understanding of S. sclerotiorum interactions with hosts of varying susceptibility levels. Here, we report for the first time on the S. sclerotiorum transcriptome during chickpea infection, which could be important for further studies on this pathogen's molecular biology.