Jean Devonshire

Molecular Mechanisms of Selenium Tolerance and Hyperaccumulation in Stanleya pinnata

The molecular mechanisms responsible for selenium (Se) tolerance and hyperaccumulation were studied in the Se hyperaccumulator Stanleya pinnata (Brassicaceae) by comparing it with the related secondary Se accumulator Stanleya albescens using a combination of physiological, structural, genomic, and biochemical approaches. S. pinnata accumulated 3.6-fold more Se and was tolerant to 20 μm selenate, while S. albescens suffered reduced growth, chlorosis and necrosis, impaired photosynthesis, and high levels of reactive oxygen species. Levels of ascorbic acid, glutathione, total sulfur, and nonprotein thiols were higher in S. pinnata, suggesting that Se tolerance may in part be due to increased antioxidants and up-regulated sulfur assimilation. S. pinnata had higher selenocysteine methyltransferase protein levels and, judged from liquid chromatography-mass spectrometry, mainly accumulated the free amino acid methylselenocysteine, while S. albescens accumulated mainly the free amino acid selenocystathionine. S. albescens leaf x-ray absorption near-edge structure scans mainly detected a carbon-Se-carbon compound (presumably selenocystathionine) in addition to some selenocysteine and selenate. Thus, S. albescens may accumulate more toxic forms of Se in its leaves than S. pinnata. The species also showed different leaf Se sequestration patterns: while S. albescens showed a diffuse pattern, S. pinnata sequestered Se in localized epidermal cell clusters along leaf margins and tips, concentrated inside of epidermal cells. Transcript analyses of S. pinnata showed a constitutively higher expression of genes involved in sulfur assimilation, antioxidant activities, defense, and response to (methyl)jasmonic acid, salicylic acid, or ethylene. The levels of some of these hormones were constitutively elevated in S. pinnata compared with S. albescens, and leaf Se accumulation was slightly enhanced in both species when these hormones were supplied. Thus, defense-related phytohormones may play an important signaling role in the Se hyperaccumulation of S. pinnata, perhaps by constitutively up-regulating sulfur/Se assimilation followed by methylation of selenocysteine and the targeted sequestration of methylselenocysteine.

Transcriptome and Metabolite Profiling of the Infection Cycle of Zymoseptoria tritici on Wheat Reveals a Biphasic Interaction with Plant Immunity Involving Differential Pathogen Chromosomal Contributions and a Variation on the Hemibiotrophic Lifestyle Definition

The temporal dynamics of Zymoseptoria tritici reproduction on Triticum aestivum involves a biphasic manipulation of plant defense responses. The hemibiotrophic fungus Zymoseptoria tritici causes Septoria tritici blotch disease of wheat (Triticum aestivum). Pathogen reproduction on wheat occurs without cell penetration, suggesting that dynamic and intimate intercellular communication occurs between fungus and plant throughout the disease cycle. We used deep RNA sequencing and metabolomics to investigate the physiology of plant and pathogen throughout an asexual reproductive cycle of Z. tritici on wheat leaves. Over 3,000 pathogen genes, more than 7,000 wheat genes, and more than 300 metabolites were differentially regulated. Intriguingly, individual fungal chromosomes contributed unequally to the overall gene expression changes. Early transcriptional down-regulation of putative host defense genes was detected in inoculated leaves. There was little evidence for fungal nutrient acquisition from the plant throughout symptomless colonization by Z. tritici, which may instead be utilizing lipid and fatty acid stores for growth. However, the fungus then subsequently manipulated specific plant carbohydrates, including fructan metabolites, during the switch to necrotrophic growth and reproduction. This switch coincided with increased expression of jasmonic acid biosynthesis genes and large-scale activation of other plant defense responses. Fungal genes encoding putative secondary metabolite clusters and secreted effector proteins were identified with distinct infection phase-specific expression patterns, although functional analysis suggested that many have overlapping/redundant functions in virulence. The pathogenic lifestyle of Z. tritici on wheat revealed through this study, involving initial defense suppression by a slow-growing extracellular and nutritionally limited pathogen followed by defense (hyper) activation during reproduction, reveals a subtle modification of the conceptual definition of hemibiotrophic plant infection.

β-Carboxysomal proteins assemble into highly organized structures in Nicotiana chloroplasts

The photosynthetic efficiency of C3 plants suffers from the reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) with O2 instead of CO2 , leading to the costly process of photorespiration. Increasing the concentration of CO2 around Rubisco is a strategy used by photosynthetic prokaryotes such as cyanobacteria for more efficient incorporation of inorganic carbon. Engineering the cyanobacterial CO2 -concentrating mechanism, the carboxysome, into chloroplasts is an approach to enhance photosynthesis or to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. We have chosen to explore the possibility of producing β-carboxysomes from Synechococcus elongatus PCC7942, a model freshwater cyanobacterium. Using the agroinfiltration technique, we have transiently expressed multiple β-carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO and CcmN) in Nicotiana benthamiana with fusions that target these proteins into chloroplasts, and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the β-carboxysome are able to assemble in plant chloroplasts into highly organized assemblies resembling empty microcompartments. We demonstrate that a foreign protein can be targeted with a 17-amino-acid CcmN peptide to the shell proteins inside chloroplasts. Our experiments establish the feasibility of introducing carboxysomes into chloroplasts for the potential compartmentalization of Rubisco or other proteins.

The Pochonia chlamydosporia Serine Protease Gene vcp1 Is Subject to Regulation by Carbon, Nitrogen and pH: Implications for Nematode Biocontrol

The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers) may enhance biocontrol potential in some circumstances.

Aberrant protein N-glycosylation impacts upon infection-related growth transitions of the haploid plant-pathogenic fungus Mycosphaerella graminicola

The ascomycete fungus Mycosphaerella graminicola is the causal agent of Septoria Tritici Blotch disease of wheat and can grow as yeast‐like cells or as hyphae depending on environmental conditions. Hyphal growth is however essential for successful leaf infection. A T‐DNA mutagenesis screen performed on haploid spores identified a mutant, which can undergo yeast‐like growth but cannot switch to hyphal growth. For this reason the mutant was non‐pathogenic towards wheat leaves. The gene affected, MgAlg2, encoded a homologue of Saccharomyces cerevisiae ScAlg2, an alpha‐1,2‐mannosyltransferase, which functions in the early stages of asparagine‐linked protein (N‐) glycosylation. Targeted gene deletion and complementation experiments confirmed that loss of MgAlg2 function prevented the developmental growth switch. MgAlg2 was able to functionally complement the S. cerevisiae ScAlg2‐1 temperature sensitive growth phenotype. Spores of ΔMgAlg2 mutants were hypersensitive to the cell wall disrupting agent Calcofluor white and produced abnormally hypo‐N‐glycosylated proteins. Gene expression, proteome and glycoproteome analysis revealed that ΔMgAlg2 mutant spores show responses typically associated with the accumulation of mis‐folded proteins. The data presented highlight key roles for protein N‐glycosylation in regulating the switch to hyphal growth, possibly as a consequence of maintaining correct folding and localization of key proteins involved in this process.

Selenium Hyperaccumulator Plants Stanleya pinnata and Astragalus bisulcatus Are Colonized by Se-Resistant, Se-Excluding Wasp and Beetle Seed Herbivores

Selenium (Se) hyperaccumulator plants can concentrate the toxic element Se up to 1% of shoot (DW) which is known to protect hyperaccumulator plants from generalist herbivores. There is evidence for Se-resistant insect herbivores capable of feeding upon hyperaccumulators. In this study, resistance to Se was investigated in seed chalcids and seed beetles found consuming seeds inside pods of Se-hyperaccumulator species Astragalus bisulcatus and Stanleya pinnata. Selenium accumulation, localization and speciation were determined in seeds collected from hyperaccumulators in a seleniferous habitat and in seed herbivores. Astragalus bisulcatus seeds were consumed by seed beetle larvae (Acanthoscelides fraterculus Horn, Coleoptera: Bruchidae) and seed chalcid larvae (Bruchophagus mexicanus, Hymenoptera: Eurytomidae). Stanleya pinnata seeds were consumed by an unidentified seed chalcid larva. Micro X-ray absorption near-edge structure (µXANES) and micro-X-Ray Fluorescence mapping (µXRF) demonstrated Se was mostly organic C-Se-C forms in seeds of both hyperaccumulators, and S. pinnata seeds contained ∼24% elemental Se. Liquid chromatography–mass spectrometry of Se-compounds in S. pinnata seeds detected the C-Se-C compound seleno-cystathionine while previous studies of A. bisulcatus seeds detected the C-Se-C compounds methyl-selenocysteine and γ-glutamyl-methyl-selenocysteine. Micro-XRF and µXANES revealed Se ingested from hyperaccumulator seeds redistributed throughout seed herbivore tissues, and portions of seed C-Se-C were biotransformed into selenocysteine, selenocystine, selenodiglutathione, selenate and selenite. Astragalus bisulcatus seeds contained on average 5,750 µg Se g−1, however adult beetles and adult chalcid wasps emerging from A. bisulcatus seed pods contained 4–6 µg Se g−1. Stanleya pinnata seeds contained 1,329 µg Se g−1 on average; however chalcid wasp larvae and adults emerging from S. pinnata seed pods contained 9 and 47 µg Se g−1. The results suggest Se resistant seed herbivores exclude Se, greatly reducing tissue accumulation; this explains their ability to consume high-Se seeds without suffering toxicity, allowing them to occupy the unique niche offered by Se hyperaccumulator plants.

Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation

Summary Peroxisomal ABC transporters have been studied extensively in Arabidopsis but not in monocotyledonous species. Using barley, it is shown that their biochemical functions are conserved in flowering plants.

Overexpression of the potassium channel TPKb in small vacuoles confers osmotic and drought tolerance to rice

Potassium (K(+) ) is the most important cationic nutrient for all living organisms. Vacuolar two-pore K(+) (TPK) channels are important players in the regulation of cellular levels of K(+) but have not been characterised in rice. In order to assess the role of OsTPKb, a K(+) selective ion channel predominantly expressed in the tonoplast of small vacuoles, we generated overexpressing (OX) lines using a constitutive promoter and compared their phenotypes with control plants. Relative to control plants, OX lines showed better growth when exposed to low-K(+) or water stress conditions. K(+) uptake was greater in OX lines which may be driven by increased AKT1 and HAK1 activity. The enhanced K(+) uptake led to tissue K(+) levels that were raised in roots and shoots. Furthermore, energy dispersive X-ray (EDX) analyses showed a higher cytoplasm: vacuole K(+) ratio which is likely to contribute to the increased stress tolerance. In all, the data suggest that TPKb can alter the K(+) status of small vacuoles, which is important for general cellular K(+) homeostasis which, in turn, affects stress tolerance.

A combined cryo-scanning electron microscopy/cryoplaning approach to study the infection of Meloidogyne incognita eggs by Pochonia chlamydosporia

The fungus Pochonia chlamydosporia is a saprophytic soil-dwelling fungus and is also a parasite of the eggs of the root-knot plant-parasitic nematodes (Meloidogyne spp.). Studies on its molecular characterisation, diversity, tritrophic interactions and ecology have been carried out. However, to elucidate the role in soil and rhizosphere ecology of this and other nematophagous fungi used in biological control, and to enhance their exploitation, it is necessary to improve the understanding of the biology and fungus-nematode infection process using different approaches, including microscopy. Low-temperature Scanning Electron Microscopy (cryo-SEM) techniques allow the examination of frozen, fully hydrated samples that can reveal important ultrastructural features occurring through fungus-nematode interactions. A method that combined cryo-SEM with cryoplaning was developed to examine samples of eggs, gelatinous matrix and females of Meloidogyne incognita colonised by P. chlamydosporia. The fungal samples were produced in potato dextrose agar to which different nematode stages were added and processed for cryo-SEM and cryoplaning within a period of 0-72 h post inoculation. The method was found to be rapid and economical, provided clear and detailed external images of the infection process and allowed viewing of sections through structures with minimal processing in comparison to other cryo-SEM techniques and it could be adapted to study other fungus-nematode interactions.

Inactivation of plant infecting fungal and viral pathogens to achieve biological containment in drainage water using UV treatment

Aim:  To explore whether ultraviolet (UV) light treatment within a closed circulating and filtered water drainage system can kill plant pathogenic species.