Marisa E. Miller

cis- and trans-Regulation of miR163 and Target Genes Confers Natural Variation of Secondary Metabolites in Two Arabidopsis Species and Their Allopolyploids

Nonadditive expression of miR163 in allopolyploids is caused largely by cis-acting promoters and by trans-acting factors present in Arabidopsis arenosa and allopolyploids but absent in Arabidopsis thaliana. miR163 negatively regulates secondary metabolite pathways in defense response. This is one example of genome-wide cis- and trans-regulation that shapes interspecific hybrids and allopolyploids. MicroRNAs (miRNAs) play essential roles in plant and animal development, but the cause and effect of miRNA expression divergence between closely related species and in interspecific hybrids or allopolyploids are unknown. Here, we show differential regulation of a miR163-mediated pathway in allotetraploids and their progenitors, Arabidopsis thaliana and Arabidopsis arenosa. miR163 is a recently evolved miRNA in A. thaliana and highly expressed in A. thaliana, but its expression was undetectable in A. arenosa and repressed in resynthesized allotetraploids. Repression of A. arenosa MIR163 (Aa MIR163) is caused by a weak cis-acting promoter and putative trans-acting repressor(s) present in A. arenosa and allotetraploids. Moreover, ectopic Aa MIR163 precursors were processed more efficiently in A. thaliana than in resynthesized allotetraploids, suggesting a role of posttranscriptional regulation in mature miR163 abundance. Target genes of miR163 encode a family of small molecule methyltransferases involved in secondary metabolite biosynthetic pathways that are inducible by a fungal elicitor, alamethicin. Loss of miR163 or overexpression of miR163 in mir163 mutant plants alters target transcript and secondary metabolite profiles. We suggest that cis- and trans-regulation of miRNA and other genes provides a molecular basis for natural variation of biochemical and metabolic pathways that are important to growth vigor and stress responses in Arabidopsis-related species and allopolyploids.

Ploidy and Hybridity Effects on Growth Vigor and Gene Expression in Arabidopsis thaliana Hybrids and Their Parents

Both ploidy and hybridity affect cell size and growth vigor in plants and animals, but the relative effects of genome dosage and hybridization on biomass, fitness, and gene expression changes have not been systematically examined. Here we performed the first comparative analysis of seed, cell, and flower sizes, starch and chlorophyll content, biomass, and gene expression changes in diploid, triploid, and tetraploid hybrids and their respective parents in three Arabidopsis thaliana ecotypes: Columbia, C24, and Landsberg erecta (Ler). Ploidy affects many morphological and fitness traits, including stomatal size, flower size, and seed weight, whereas hybridization between the ecotypes leads to altered expression of central circadian clock genes and increased starch and chlorophyll content, biomass, and seed weight. However, varying ploidy levels has subtle effects on biomass, circadian clock gene expression, and chlorophyll and starch content. Interestingly, biomass, starch content, and seed weight are significantly different between the reciprocal hybrids at all ploidy levels tested, with the lowest and highest levels found in the reciprocal triploid hybrids, suggesting parent-of-origin effects on biomass, starch content, and seed weight. These findings provide new insights into molecular events of polyploidy and heterosis, as well as complex agronomic traits that are important to biomass and seed production in hybrid and polyploid crops.

Proteomic divergence in Arabidopsis autopolyploids and allopolyploids and their progenitors

Autopolyploidy and allopolyploidy are common in many plants and some animals. Rapid changes in genomic composition and gene expression have been observed in both autopolyploids and allopolyploids, but the effects of polyploidy on proteomic divergence are poorly understood. Here, we report quantitative analysis of protein changes in leaves of Arabidopsis autopolyploids and allotetraploids and their progenitors using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. In more than 1000 proteins analyzed, the levels of protein divergence were relatively high (∼18%) between Arabidopsis thaliana and Arabidopsis arenosa, relatively low (∼6.8%) between an A. thaliana diploid and autotetraploid and intermediate (∼8.3 and 8.2%) in F1- and F8-resynthesized allotetraploids relative to mid-parent values, respectively. This pattern of proteomic divergence was consistent with the previously reported gene expression data. In particular, many non-additively accumulated proteins (61–62%) in the F1 and F8 allotetraploids were also differentially expressed between the parents. The differentially accumulated proteins in functional categories of abiotic and biotic stresses were overrepresented between an A. thaliana autotetraploid and diploid and between two Arabidopsis species, but not significantly different between allotetraploids and their progenitors. Although the trend of changes is similar, the percentage of differentially accumulated proteins that matched previously reported differentially expressed genes was relatively low. Western blot analysis confirmed several selected proteins with isoforms the cumulative levels of which were differentially expressed. These data suggest high protein divergence between species and rapid changes in post-transcriptional regulation and translational modifications of proteins during polyploidization.

Natural variation in timing of stress-responsive gene expression predicts heterosis in intraspecific hybrids of Arabidopsis

The genetic distance between hybridizing parents affects heterosis; however, the mechanisms for this remain unclear. Here we report that this genetic distance correlates with natural variation and epigenetic regulation of circadian clock-mediated stress responses. In intraspecific hybrids of Arabidopsis thaliana, genome-wide expression of many biotic and abiotic stress-responsive genes is diurnally repressed and this correlates with biomass heterosis and biomass quantitative trait loci. Expression differences of selected stress-responsive genes among diverse ecotypes are predictive of heterosis in their hybrids. Stress-responsive genes are repressed in the hybrids under normal conditions but are induced to mid-parent or higher levels under stress at certain times of the day, potentially balancing the tradeoff between stress responses and growth. Consistent with this hypothesis, repression of two candidate stress-responsive genes increases growth vigour. Our findings may therefore provide new criteria for effectively selecting parents to produce high- or low-yield hybrids.

A Role for CHH Methylation in the Parent-of-Origin Effect on Altered Circadian Rhythms and Biomass Heterosis in Arabidopsis Intraspecific Hybrids

This work describes a unique role for the RNA-directed DNA methylation pathway (mainly CHH methylation, where H = A, T, or C) in mediating the parent-of-origin effect on the expression of the circadian clock gene CCA1 in Arabidopsis intraspecific hybrids. Altered CCA1 expression amplitudes are associated with heterosis of embryo growth and biomass accumulation in the reciprocal hybrids. Hybrid plants and animals often show increased levels of growth and fitness, a phenomenon known as hybrid vigor or heterosis. Circadian rhythms optimize physiology and metabolism in plants and animals. In plant hybrids and polyploids, expression changes of the genes within the circadian regulatory network, such as CIRCADIAN CLOCK ASSOCIATED1 (CCA1), lead to heterosis. However, the relationship between allelic CCA1 expression and heterosis has remained elusive. Here, we show a parent-of-origin effect on altered circadian rhythms and heterosis in Arabidopsis thaliana F1 hybrids. This parent-of-origin effect on biomass heterosis correlates with altered CCA1 expression amplitudes, which are associated with methylation levels of CHH (where H = A, T, or C) sites in the promoter region. The direction of rhythmic expression and hybrid vigor is reversed in reciprocal F1 crosses involving mutants that are defective in the RNA-directed DNA methylation pathway (argonaute4 and nuclear RNA polymerase D1a) but not in the maintenance methylation pathway (methyltransferase1 and decrease in DNA methylation1). This parent-of-origin effect on circadian regulation and heterosis is established during early embryogenesis and maintained throughout growth and development.

The role of mitochondria in plant development and stress tolerance

Eukaryotic cells require orchestrated communication between nuclear and organellar genomes, perturbations in which are linked to stress response and disease in both animals and plants. In addition to mitochondria, which are found across eukaryotes, plant cells contain a second organelle, the plastid. Signaling both among the organelles (cytoplasmic) and between the cytoplasm and the nucleus (i.e. nuclear-cytoplasmic interactions (NCI)) is essential for proper cellular function. A deeper understanding of NCI and its impact on development, stress response, and long-term health is needed in both animal and plant systems. Here we focus on the role of plant mitochondria in development and stress response. We compare and contrast features of plant and animal mitochondrial genomes (mtDNA), particularly highlighting the large and highly dynamic nature of plant mtDNA. Plant-based tools are powerful, yet underutilized, resources for enhancing our fundamental understanding of NCI. These tools also have great potential for improving crop production. Across taxa, mitochondria are most abundant in cells that have high energy or nutrient demands as well as at key developmental time points. Although plant mitochondria act as integrators of signals involved in both development and stress response pathways, little is known about plant mtDNA diversity and its impact on these processes. In humans, there are strong correlations between particular mitotypes (and mtDNA mutations) and developmental differences (or disease). We propose that future work in plants should focus on defining mitotypes more carefully and investigating their functional implications as well as improving techniques to facilitate this research.

De Novo Assembly and Phasing of Dikaryotic Genomes from Two Isolates of Puccinia coronata f. sp. avenae, the Causal Agent of Oat Crown Rust

ABSTRACT Oat crown rust, caused by the fungus Pucinnia coronata f. sp. avenae, is a devastating disease that impacts worldwide oat production. For much of its life cycle, P. coronata f. sp. avenae is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two P. coronata f. sp. avenae isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for 12SD80, for a total assembly length of 99.16 Mbp, and 777 primary contigs for 12NC29, for a total length of 105.25 Mbp; approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of coexpressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in P. coronata f. sp. avenae. IMPORTANCE Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae, which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding P. coronata f. sp. avenae, resources to study the molecular mechanisms underpinning pathogenicity and the emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of P. coronata f. sp. avenae as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of P. coronata f. sp. avenae. IMPORTANCE Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae, which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding P. coronata f. sp. avenae, resources to study the molecular mechanisms underpinning pathogenicity and the emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of P. coronata f. sp. avenae as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of P. coronata f. sp. avenae.

A Near-Complete Haplotype-Phased Genome of the Dikaryotic Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici Reveals High Interhaplotype Diversity

ABSTRACT A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales. In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies. IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organism’s evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis. IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organism’s evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.

Identification, introgression, and molecular marker genetic analysis and selection of a highly effective novel oat crown rust resistance from diploid oat, Avena strigosa

Key messageOat crown rust is one of the most damaging diseases of oat. We identified a new source of resistance and developed KASP and TaqMan markers for selection in breeding programs.AbstractA new highly effective resistance to oat crown rust (Puccinia coronata f. sp. avenae) was identified in the diploid oat Avena strigosa PI 258731 and introgressed into hexaploid cultivated oat. Young plants with this resistance show moderate susceptibility, whereas older plant tissues and adult plants are resistant with no virulent isolates encountered in over 8xa0years of testing. Resistance was incorporated into hexaploid oat by embryo rescue, colchicine chromosome doubling followed by backcrosses with a hexaploid parent, and selection for stable transmission of resistance. To mitigate flag leaf and panicle chlorosis/necrosis associated with the resistance, crosses were made with derived resistant lines to breeding lines of divergent parentage followed by selection. Subsequently, two F2 sister lines, termed MNBT1020-1 and MNBT1021-1, were identified in which the chlorosis/necrosis was reduced. These two lines performed well in replicated multi-location state trials in 2015 and 2016 out-yielding all cultivar entries. Segregating F2:3 plants resulting from crosses of MNBT lines to susceptible parents were genotyped with the oat 6K SNP array, and SNP loci with close linkage to the resistance were identified. KASP assays generated from linked SNPs showed accurate discrimination of the resistance in derivatives of the resistant MNBT lines crossed to susceptible breeding lines. A TaqMan marker was developed and correctly identified homozygous resistance in over 95% of 379 F4 plants when rust was scored in F4:5 plants in the field. Thus, a novel highly effective resistance and associated molecular markers are available for use in breeding, genetic analysis, and functional studies.

A Novel Approach to Assess Salt Stress Tolerance in Wheat Using Hyperspectral Imaging

Salinity stress has significant adverse effects on crop productivity and yield. The primary goal of this study was to quantitatively rank salt tolerance in wheat using hyperspectral imaging. Four wheat lines were assayed in a hydroponic system with control and salt treatments (0 and 200 mM NaCl). Hyperspectral images were captured one day after salt application when there were no visual symptoms. Subsequent to necessary preprocessing tasks, two endmembers, each representing one of the treatment, were identified in each image using successive volume maximization. To simplify image analysis and interpretation, similarity of all pixels to the salt endmember was calculated by a technique proposed in this study, referred to as vector-wise similarity measurement. Using this approach allowed high-dimensional hyperspectral images to be reduced to one-dimensional gray-scale images while retaining all relevant information. Two methods were then utilized to analyze the gray-scale images: minimum difference of pair assignments and Bayesian method. The rankings of both methods were similar and consistent with the expected ranking obtained by conventional phenotyping experiments and historical evidence of salt tolerance. This research highlights the application of machine learning in hyperspectral image analysis for phenotyping of plants in a quantitative, interpretable, and non-invasive manner.