Parwinder Kaur

Decreasing Electron Flux through the Cytochrome and/or Alternative Respiratory Pathways Triggers Common and Distinct Cellular Responses Dependent on Growth Conditions

Diverse signaling pathways are activated by perturbation of mitochondrial function under different growth conditions. Diverse signaling pathways are activated by perturbation of mitochondrial function under different growth conditions.Mitochondria have emerged as an important organelle for sensing and coping with stress in addition to being the sites of important metabolic pathways. Here, responses to moderate light and drought stress were examined in different Arabidopsis (Arabidopsis thaliana) mutant plants lacking a functional alternative oxidase (alternative oxidase1a [aox1a]), those with reduced cytochrome electron transport chain capacity (T3/T7 bacteriophage-type RNA polymerase, mitochondrial, and plastidial [rpoTmp]), and double mutants impaired in both pathways (aox1a:rpoTmp). Under conditions considered optimal for growth, transcriptomes of aox1a and rpoTmp were distinct. Under adverse growth conditions, however, transcriptome changes in aox1a and rpoTmp displayed a highly significant overlap and were indicative of a common mitochondrial stress response and down-regulation of photosynthesis. This suggests that the role of mitochondria to support photosynthesis is provided through either the alternative pathway or the cytochrome pathway, and when either pathway is inhibited, such as under environmental stress, a common, dramatic, and succinct mitochondrial signal is activated to alter energy metabolism in both organelles. aox1a:rpoTmp double mutants grown under optimal conditions showed dramatic reductions in biomass production compared with aox1a and rpoTmp and a transcriptome that was distinct from aox1a or rpoTmp. Transcript data indicating activation of mitochondrial biogenesis in aox1a:rpoTmp were supported by a proteomic analysis of over 200 proteins. Under optimal conditions, aox1a:rpoTmp plants seemed to switch on many of the typical mitochondrial stress regulators. Under adverse conditions, aox1a:rpoTmp turned off these responses and displayed a biotic stress response. Taken together, these results highlight the diverse signaling pathways activated by the perturbation of mitochondrial function under different growth conditions.

Genetic improvement of subterranean clover (Trifolium subterraneum L.). 1. Germplasm, traits and future prospects

Abstract. Subterranean clover (Trifolium subterraneum L.) is the most widely sown annual pasture legume species in southern Australia, valued in the livestock and grains industries as a source of high-quality forage and for its ability to fix atmospheric nitrogen. From its initial accidental introduction into Australia in the 19th Century and subsequent commercialisation in the early 1900s, 45 cultivars have been registered in Australia. These consist of 32 cultivars of ssp. subterraneum, eight of ssp. yanninicum, and five of ssp. brachycalycinum and range in flowering time from 77 to 163 days from sowing, enabling the species to be grown in a diversity of rainfall environments, soil types, and farming systems. Eleven of these cultivars are introductions from the Mediterranean region, 15 are naturalised strains collected in Australia, 18 are the products of crossbreeding, and one is derived from mutagenesis. Cultivars developed in Italy have been commercialised for the local market, whereas other cultivars developed in Spain, Portugal, and France have not had commercial seed production. Important traits exploited include: (i) selection for low levels of the oestrogenic isoflavone formononetin, which causes reduced ewe fertility; (ii) increased levels of dormancy imposed by seed-coat impermeability (hard seeds) for cultivars aimed at crop rotations or unreliable rainfall environments; (iii) strong burr-burial ability to maximise seed production; (iv) resistance to important disease pathogens for cultivars aimed at medium- and high-rainfall environments, particularly to Kabatiella caulivora and root rot pathogens; (v) resistance to pests, particularly redlegged earth mites; and (vi) selection for unique leaf markings and other morphological traits (where possible) to aid cultivar identification. Cultivar development has been aided by a large genetic resource of ∼10 000 accessions, assembled from its centre of origin in the Mediterranean Basin, West Asia, and the Atlantic coast of Western Europe, in addition to naturalised strains collected in Australia. The development of a core collection of 97 accessions, representing almost 80% of the genetic diversity of the species, and a genetic map, provides a platform for development of future cultivars with new traits to benefit the livestock and grains industries. New traits being examined include increased phosphorous-use efficiency and reduced methane emissions from grazing ruminant livestock. Economic analyses indicate that future trait development should focus on traits contributing to increased persistence and autumn–winter productivity, while other potential traits include increased nutritive value (particularly of senesced material), increased N2 fixation ability, and tolerance to cheap herbicides. Beneficial compounds for animal and human health may also be present within the species for exploitation.

Proteome analysis of the Albugo candida–Brassica juncea pathosystem reveals that the timing of the expression of defence-related genes is a crucial determinant of pathogenesis

White rust, caused by Albugo candida, is a serious pathogen of Brassica juncea (Indian mustard) and poses a potential hazard to the presently developing canola-quality B. juncea industry worldwide. A comparative proteomic study was undertaken to explore the molecular mechanisms that underlie the defence responses of Brassica juncea to white rust disease caused by the biotrophic oomycete Albugo candida. Nineteen proteins showed reproducible differences in abundance between a susceptible (RH 819) and a resistant variety (CBJ 001) of B. juncea following inoculation with A. candida. The identities of all 19 proteins were successfully established through Q-TOF MS/MS. Five of these proteins were only detected in the resistant variety and showed significant differences in their abundance at various times following pathogen inoculation in comparison to mock-inoculated plants. Among these was a thaumatin-like protein (PR-5), a protein not previously associated with the resistance of B. juncea towards A. candida. One protein, peptidyl-prolyl cis/trans isomerase (PPIase) isoform CYP20-3, was only detected in the susceptible variety and increased in abundance in response to the pathogen. PPIases have recently been discovered to play an important role in pathogenesis by suppressing the host cells immune response. For a subset of seven proteins examined in more detail, an increase in transcript abundance always preceded their induction at the proteome level. These findings are discussed within the context of the A. candida–Brassica juncea pathosystem, especially in relation to host resistance to this pathogen.

Pathogenic behaviour of strains of Albugo candida from Brassica juncea (Indian mustard) and Raphanus raphanistrum (wild radish) in Western Australia

White rust caused by Albugo candida is a serious pathogen of Brassica juncea (Indian mustard) and one posing a potential hazard to the presently developing canola-quality B. juncea industry in Australia. The varieties of B. juncea available in Australia are known to be susceptible to attack by race 2 of A. candida, a race that is known to have two pathotypes, 2A and 2V. Two isolates of A. candida, representing strains collected from B. juncea and Raphanus raphanistrum (wild radish) from two sites in Western Australia (WA) were tested on cruciferous host differentials to characterise their pathogenic behaviour. The results clearly showthat the strains obtained from B. juncea and R. raphanistrum are different in their host range. The isolate from B. juncea showed significant levels of sporulation on both differential cultivars of B. juncea, Vulcan and Commercial Brown, used for differentiating pathotype 2A from 2V, confirming the presence of pathotype 2V in WA. This is the first report of pathotype 2V in Australia. This same isolate was able to infect Brassica napus from China (FAN 189), B. tournefortii (wild turnip) B. nigra and R. sativus. This serves as a caution to breeders when sourcing resistance against A. candida from B. napus germplasm. The isolate from R. raphanistrum, tested against the same set of cruciferous host differentials, caused significant sporulation on B. juncea differential Commercial Brown, B. napus (FAN 189), B. nigra (90745), R. raphanistrum and R. sativus (White Icicle). Our strain from R. raphanistrum, while being a direct threat to B. juncea, may be a hazard, not only to any B. napus germplasm developed from B. napus breeding lines from China, but also to B. nigra and R. sativus, should these species be utilised commercially in Australia. The race delineations of these strains from the WA grainbelt are expected to be helpful for selecting appropriate genotype resistance(s) for sowing in relation to the planned expansion of the B. juncea industry and also if there is future development of alternative cruciferous oilseed industries in WA.

Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts

Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility to R. solani when expressed in Nicotiana benthamiana. In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806.

An advanced reference genome of Trifolium subterraneum L. reveals genes related to agronomic performance

Summary Subterranean clover is an important annual forage legume, whose diploidy and inbreeding nature make it an ideal model for genomic analysis in Trifolium. We reported a draft genome assembly of the subterranean clover TSUd_r1.1. Here we evaluate genome mapping on nanochannel arrays and generation of a transcriptome atlas across tissues to advance the assembly and gene annotation. Using a BioNano‐based assembly spanning 512 Mb (93% genome coverage), we validated the draft assembly, anchored unplaced contigs and resolved misassemblies. Multiple contigs (264) from the draft assembly coalesced into 97 super‐scaffolds (43% of genome). Sequences longer than >1 Mb increased from 40 to 189 Mb giving 1.4‐fold increase in N50 with total genome in pseudomolecules improved from 73 to 80%. The advanced assembly was re‐annotated using transcriptome atlas data to contain 31 272 protein‐coding genes capturing >96% of the gene content. Functional characterization and GO enrichment confirmed gene expression for response to water deprivation, flavonoid biosynthesis and embryo development ending in seed dormancy, reflecting adaptation to the harsh Mediterranean environment. Comparative analyses across Papilionoideae identified 24 893 Trifolium‐specific and 6325 subterranean‐clover‐specific genes that could be mined further for traits such as geocarpy and grazing tolerance. Eight key traits, including persistence, improved livestock health by isoflavonoid production in addition to important agro‐morphological traits, were fine‐mapped on the high‐density SNP linkage map anchored to the assembly. This new genomic information is crucial to identify loci governing traits allowing marker‐assisted breeding, comparative mapping and identification of tissue‐specific gene promoters for biotechnological improvement of forage legumes.

Draft genome sequence of subterranean clover, a reference for genus Trifolium

Clovers (genus Trifolium) are widely cultivated across the world as forage legumes and make a large contribution to livestock feed production and soil improvement. Subterranean clover (T. subterraneum L.) is well suited for genomic and genetic studies as a reference species in the Trifolium genus, because it is an annual with a simple genome structure (autogamous and diploid), unlike the other economically important perennial forage clovers, red clover (T. pratense) and white clover (T. repens). This report represents the first draft genome sequence of subterranean clover. The 471.8 Mb assembled sequence covers 85.4% of the subterranean clover genome and contains 42,706 genes. Eight pseudomolecules of 401.1 Mb in length were constructed, based on a linkage map consisting of 35,341 SNPs. The comparative genomic analysis revealed that different clover chromosomes showed different degrees of conservation with other Papilionoideae species. These results provide a reference for genetic and genomic analyses in the genus Trifolium and new insights into evolutionary divergence in Papilionoideae species.

First Report of Powdery Mildew Caused by Erysiphe cruciferarum on Brassica juncea in Australia

In Australia, Brassica juncea (L.) Czern & Coss (Indian mustard) has the potential as a more drought-tolerant oilseed crop than the B. napus L., with the first canola-quality B. juncea varieties released in Australia in 2006 and first sown for commercial production in 2007. Increased production of B. juncea is expected to result in the appearance of diseases previously unreported in Australia. In the spring of 2007 at the University of Western Australia field plots at Crawley (31.99°S, 115.82°E), Western Australia, plants of B. juncea genotypes from Australia and China had extensive stem colonization by powdery mildew at the end of the flowering period, with whitish patches ranging in size from 3 mm to 3 cm long. These patches coalesced to form a dense, white, powdery layer as they expanded. Pathogenicity was demonstrated by gently pressing infected stems containing abundant sporulation onto leaves of potted B. juncea seedlings of variety JM-18, incubating the plants in a moist chamber for 48 h, and then maintaining the plants in a controlled-environment room at 18/13°C for day/night. Signs of powdery mildew appeared at 7 days after inoculation, and by 10 days, it was well developed. Uninoculated control plants did not have powdery mildew. When symptomatic plants were examined, abundant conidia were typical of Erysiphe cruciferarum Opiz ex Junell, with cylindrical conidia borne singly or in short chains as described previously (2). Mycelia were amphigenous, in patches, and often spreading to become effused. Conidiophores were straight, foot cells were cylindrical, and conidia were mostly produced singly and measured 21.2 to 35.4 (mean 26.7 μm) × 8.8 to 15.9 μm (mean 11.9 μm) from measurements of 100 conidia. The spore size that we measured approximated what was found for E. cruciferarum (2) (30 to 40 × 12 to 16 μm), since we found 35 and 50% of spores falling within this range in terms of length and width, respectively. Conidia were, however, generally smaller in size than that reported on broccoli raab in California (1) (35 to 50 × 12 to 21 μm). We confirmed a length-to-width ratio greater than 2 as was found previously (1,2). Infected leaves showed signs of early senescence. While powdery mildew caused by E. cruciferarum is an important disease of B. juncea in India where yield losses as much as 17% have been reported (4), its potential impact in Australia is yet to be determined. To our knowledge, this is the first record of E. cruciferarum on B. juncea in Australia. In Western Australia, E. cruciferarum has been recorded on B. napus (oilseed rape) since 1986 and on B. napus L. var. napobrassica (L.) Reichenb. (swede) since 1971 (3). In other regions of Australia, it has been recorded on B. rapa in Queensland since 1913 and on B. napus (oilseed rape) in South Australia since 1973. References: (1) S. T. Koike and G. S. Saenz. Plant Dis. 81:1093, 1997. (2) T. J. Purnell and A. Sivanesan. No 251 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1970. (3) R. G. Shivas. J. R. Soc. West. Aust. 72:1, 1989. (4) A. K. Shukla et al. Manual on Management of Rapeseed-Mustard Diseases. National Research Centre on Rapeseed-Mustard, Bharatpur, India, 2003.

Large-scale structural variation detection in subterranean clover subtypes using optical mapping validated at nucleotide level

Whole genome sequencing has been widely used to detect structural variations (SVs). However, the limited single molecule size makes it difficult to characterize large-scale SVs in a genome because they cannot fully cover such vast and complex regions. Recently, optical mapping in nanochannels has provided novel resolution to detect large-scale SVs by comparing the physical location of the nickase recognition sequence in genomes. Other than in humans, SVs discovered in plants by optical mapping have not been validated. To assess the accuracy of SV calling in plants by optical mapping, we selected two genetically diverse subspecies of the Trifolium model species, subterranean clover cvs. Daliak and Yarloop. The SVs discovered by BioNano optical mapping (BOM) were validated using Illumina short reads. In the analysis, BOM identified 12 large-scale regions containing deletions and 19 containing insertions in Yarloop. The 12 large-scale regions contained 71 small deletions when validated by Illumina short reads. The results suggest that BOM could detect the total size of deletions and insertions, but it could not precisely report the location and actual quantity of SVs in the genome. Nucleotide-level validation is crucial to confirm and characterize SVs reported by optical mapping. The accuracy of SV detection by BOM is highly dependent on the quality of reference genomes and the density of selected nickases.

Climate clever clovers: New paradigm to reduce the environmental footprint of ruminants by breeding low methanogenic forages utilizing haplotype variation

Mitigating methane production by ruminants is a significant challenge to global livestock production. This research offers a new paradigm to reduce methane emissions from ruminants by breeding climate-clever clovers. We demonstrate wide genetic diversity for the trait methanogenic potential in Australia’s key pasture legume, subterranean clover (Trifolium subterraneum L.). In a bi-parental population the broadsense heritability in methanogenic potential was moderate (H2 = 0.4) and allelic variation in a region of Chr 8 accounted for 7.8% of phenotypic variation. In a genome-wide association study we identified four loci controlling methanogenic potential assessed by an in vitro fermentation system. Significantly, the discovery of a single nucleotide polymorphism (SNP) on Chr 5 in a defined haplotype block with an upstream putative candidate gene from a plant peroxidase-like superfamily (TSub_g18548) and a downstream lectin receptor protein kinase (TSub_g18549) provides valuable candidates for an assay for this complex trait. In this way haplotype variation can be tracked to breed pastures with reduced methanogenic potential. Of the quantitative trait loci candidates, the DNA-damage-repair/toleration DRT100-like protein (TSub_g26967), linked to avoid the severity of DNA damage induced by secondary metabolites, is considered central to enteric methane production, as are disease resistance (TSub_g26971, TSub_g26972, and TSub_g18549) and ribonuclease proteins (TSub_g26974, TSub_g26975). These proteins are good pointers to elucidate the genetic basis of in vitro microbial fermentability and enteric methanogenic potential in subterranean clover. The genes identified allow the design of a suite of markers for marker-assisted selection to reduce rumen methane emission in selected pasture legumes. We demonstrate the feasibility of a plant breeding approach without compromising animal productivity to mitigate enteric methane emissions, which is one of the most significant challenges to global livestock production.