Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing which bypasses embryo lethality.

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate cross-linking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect cross-linking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here, we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MGP2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not cross-link normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→sidechain). We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II

Functional characterisation of plant cytosolic thioredoxins.

Thioredoxins are small, ubiquitous, disulfide oxidoreductase proteins characterised by a conserved dicysteine active site. Within the cell, they are believed to maintain the redox environment and participate in a broad range of biochemical processes. Plant thioredoxins are a diverse multigene family, primarily classified according to the system by which they are reduced and their subcellular localization. Thioredoxins located in the cytoplasm (type -h) are usually dependent on NADPH for reduction by NADPH-thioredoxin reductase. There are four cytosolic thioredoxins in grass species, with subclass 4 believed to be the most ancient. The highly conserved nature of thioredoxin-h4, in plant species as diverse as angiosperms and gymnosperms, implies a conservation of gene function. Discovery of thioredoxin-h4 function in barley (Hordeum vulgare) was the core focus of the research presented in this thesis. The characterisation of thioredoxin-h4 was approached from both, genetic, and protein biochemistry perspectives. To commence the research, the transcript profile of barley thioreodoxin-h4 (HvTrx-h4) was examined in barley reproductive tissues. As a direct consequence of findings, anther and stigma tissues were used in protein interaction studies employing a mono-cystenic active-site HvTrx-h4 affinity chromatography technique. HvTrx-h4 was mutated, recombinantly expressed, purified and immobilised in order to isolate and identify proteins with which it interacted. Identification of HvTrx-h4 protein targets sought to reveal the pathways in which thioredoxin-h4 is involved. To further characterise the expression of HvTrx-h4, the promoter and 5′ untranslated regions of genomic sequence were isolated and used to drive expression of green fluorescent protein in transgenically modified barley. This enabled examination of the temporal and spatial regulation of HvTrx-h4 under normal growth conditions, as well as in response to abiotic stress and plant hormone treatments. Through these studies it was discovered that HvTrx-h4 is likely to be the subject of post-transcriptional modifications. Subsequent investigations revealed HvTrx-h4 is also regulated at the post-translational level through glutathionylation. Previous studies have ascribed a role for thioredoxins in plant oxidative stress defence. The question of whether modulation of HvTrx-h4 expression could be manipulated to alter plant oxidative stress tolerance was considered. To investigate, transgenic tobacco plants (Nicotiana tabacum) containing altered amounts of thioredoxin-h4 protein were subjected to various stresses; abiotic, biotic and chemical, in nature. Tobacco constitutively over-expressing thioredoxin-h4 displayed increased tolerance to ultraviolet light B, heat and methyl viologen treatment. Knowledge acquired by this study and presented in this thesis, suggest a role for barley thioredoxin-h4 in the oxidative stress response. Furthermore, the description of both post-transcriptional and post-translational regulation of HvTrx-h4 constitutes the first report of this level of regulation for a plant cytosolic thioredoxin.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201

Multiple resistance to acetohydroxyacid synthase-inhibiting and auxinic herbicides in a population of oriental mustard (Sisymbrium orientale)

A population of oriental mustard from Port Broughton in South Australia was reported as not being controlled by 2,4-D. Dose response experiments determined this population was resistant to both 2,4-D and MCPA, requiring greater than 20 times more herbicide for equivalent control compared to a known susceptible population (from Roseworthy, South Australia) and a population resistant only to the acetohydroxyacid synthase (AHAS)-inhibiting herbicides (from Tumby Bay, South Australia). The Port Broughton population was also found to be resistant to three chemical groups that inhibit AHAS; however, the level of resistance was lower than the known acetolactate synthase–resistant population from Tumby Bay. Herbicides from other modes of action were able to control the Port Broughton population. Assays of isolated AHAS from the Port Broughton population showed high levels of resistance to the sulfonylurea and sulfonamide herbicide groups, but not to the imidazolinone herbicides. A single nucleotide change in the AHAS gene that predicted a Pro to Ser substitution at position 197 in the protein was identified in the Port Broughton population. This population of oriental mustard has evolved multiple resistance to AHAS-inhibiting herbicides (AHAS inhibitors) and auxinic herbicides, through a mutation in AHAS and a second nontarget-site mechanism. Whether the same mechanism provides resistance to both AHAS inhibitors and auxinic herbicides remains to be determined. Multiple resistance to auxinic herbicides and AHAS inhibitors in the Port Broughton population will make control of this population more difficult.Nomenclature: 2,4-D; MCPA; oriental mustard, Sisymbrium orientale Torn.Christopher Preston, Fleur C. Dolman, and Peter Boutsali

A decade of glyphosate-resistant Lolium around the world: Mechanisms, genes, fitness, and agronomic management

Copyright © 2009 BioOne All rights reservedGlyphosate resistance was first discovered in populations of rigid ryegrass in Australia in 1996. Since then, glyphosate resistance has been detected in additional populations of rigid ryegrass and Italian ryegrass in several other countries. Glyphosate-resistant rigid ryegrass and Italian ryegrass have been selected in situations where there is an overreliance on glyphosate to the exclusion of other weed control tactics. Two major mechanisms of glyphosate resistance have been discovered in these two species: a change in the pattern of glyphosate translocation such that glyphosate accumulates in the leaf tips of resistant plants instead of in the shoot meristem; and amino acid substitutions at Pro 106 within the target site, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). There are also populations with both mechanisms. In the case of glyphosate resistance, the target site mutations tend to provide a lower level of resistance than does the altered translocation mechanism. Each of these resistance mechanisms is inherited as a single gene trait that is largely dominant. As these ryegrass species are obligate outcrossers, this ensures resistance alleles can move in both pollen and seed. Some glyphosate-resistant rigid ryegrass populations appear to have a significant fitness penalty associated with the resistance allele. Field surveys show that strategies vary in their ability to reduce the frequency of glyphosate resistance in populations and weed population size, with integrated strategies—including alternative weed management and controlling seed set of surviving plants—the most effective. Nomenclature: Glyphosate; rigid ryegrass, Lolium rigidum Gaud. LOLRI; Italian ryegrass, Lolium multiflorum Lam. LOLMU.Christopher Preston, Angela M. Wakelin, Fleur C. Dolman, Yazid Bostamam, and Peter Boutsali

Rigid ryegrass (Lolium rigidum) populations containing a target site mutation in EPSPS and reduced glyphosate translocation are more resistant to glyphosate

Glyphosate is widely used for weed control in the grape growing industry in southern Australia. The intensive use of glyphosate in this industry has resulted in the evolution of glyphosate resistance in rigid ryegrass. Two populations of rigid ryegrass from vineyards, SLR80 and SLR88, had 6- to 11-fold resistance to glyphosate in dose-response studies. These resistance levels were higher than two previously well-characterized glyphosate-resistant populations of rigid ryegrass (SLR77 and NLR70), containing a modified target site or reduced translocation, respectively. Populations SLR80 and SLR88 accumulated less glyphosate, 12 and 17% of absorbed glyphosate, in the shoot in the resistant populations compared with 26% in the susceptible population. In addition, a mutation within the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) where Pro106 had been substituted by either serine or threonine was identified. These two populations are more highly resistant to glyphosate as a consequence of expressing two different resistance mechanisms concurrently. Nomenclature: Glyphosate; rigid ryegrass, Lolium rigidum Gaud. LOLRI.Yazid Bostamam, Jenna M. Malone, Fleur C. Dolman, Peter Boutsalis, and Christopher Presto

Structural basis for RNA-genome recognition during bacteriophage Q replication

Upon infection of Escherichia coli by bacteriophage Q, the virus-encoded -subunit recruits host trans-lation elongation factors EF-Tu and EF-Ts and riboso-mal protein S1 to form the Q replicase holoenzyme complex, which is responsible for amplifying the Q (+)-RNA genome. Here, we use X-ray crystallogra-phy, NMR spectroscopy, as well as sequence con-servation, surface electrostatic potential and muta-tional analyses to decipher the roles of the -subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1–2) in the recognition of Q (+)-RNA by the Q replicase complex. We show how three basic residues of the subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Q replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the -subunit and protein S1 cooperatively bind the (+)-stranded Q genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation