Targeting Cell Wall Formation in the Oomycete Phytophthora cinnamomi for Disease Control

The oomycete Phytophthora genus comprises microorganisms that cause devastating plant diseases, such as late blight and root rot diseases, leading to significant agricultural economic losses, and causing extensive damages to ecosystems. To date, no practical method is available to prevent these diseases. Furthermore, current strategies to control Phytophthora-induced diseases are ineffective in the long term. These strategies currently rely on different classes of chemicals. However, repeated use of the same chemicals can lead to development of pesticide resistance in phytopathogens. This concern, combined with an increased awareness of alternative approaches that have minimal impact on biodiversity and human health, highlights that efficient methods for controlling diseases caused by Phytophthora are urgently required. Targeting cell wall biosynthesis is a promising strategy to combat these pathogens. Indeed, the inhibition of enzymes involved in carbohydrate biosynthesis affects the growth and survival of these pathogens, offering a promising avenue for the development of effective treatments. In recent years, plant antimicrobial peptides (AMPs) have been found to be effective against different phytopathogens. Well-known AMPs are plant defensins, a family of small cysteinerich peptides that can bind to chitin and cell wall glucans in fungi. However, knowledge about the inhibitory role of plant defensins in oomycetes is limited. As such, this work investigates the effects of the plant defensin NaD1 (Nicotiana alata defensin 1) on Phytophthora species, which may reveal novel opportunities for controlling plant diseases. Our findings demonstrate that NaD1 effectively inhibits the mycelial growth of Phytophthora cinnamomi, Phytophthora cambivora, Phytophthora nicotianae, and Phytophthora citricola. Exposure to NaD1 induced alterations in the growth and structure of P. cinnamomi, leading to suppressed apical dominance, hyper-branching, and changes in cell wall composition, likely due to disruption of calcium homeostasis. Transcriptomic analyses confirmed altered expression of genes involved in cellulose synthesis and calcium transport (Chapter 2), and uncovered changes in the transcriptome across the entire genome in hyphal cells exposed to NaD1, shedding light on the mechanism of action of this AMP. These differentially expressed genes can serve as candidates to study the efficacy of NaD1 against Phytophthora species (Chapter 4). In addition to NaD1, the effects of a chitin synthase inhibitor, nikkomycin Z, were also investigated. This study shows that nikkomycin Z causes strong growth inhibition of four Phytophthora species and induces abnormal hyphal growth. Exposure to this inhibitor decreases cellulose levels and affects the expression of genes related to vital functions such as cell wall biosynthesis, hexosamine biosynthesis and chitin deacetylation (Chapter 3). Altogether, the present work reveals critical information about the fundamental inhibitory mechanisms of NaD1 and nikkomycin Z on Phytophthora species, with a focus on cell wall biosynthesis. This work paves the way for the development of novel effective targets for oomycete disease control.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 202

Twentieth Fungal Genetics Conference Scientific Program

Abstracts are published as an electronic supplement to Fungal Genetics Newsletter # 46 and should be cited as follows: Fungal Genet. Newsl. 46S: Abstract Numbe

Abstracts from the 11th European Conference on Fungal Genetics

Programs and Abstracts from the 11th European Conference on Fungal Genetic

Molecular characterization of the Arabidopsis thaliana - Botrytis cinerea interaction

Includes bibliographical references (leaves 199-253).This study attempted to characterize at a transcriptional level, the defence responses of Arabidopsis thaliana after infection by Botrytis cinerea, using microarrays. The first microarray experiment focused on profiling Arabidopsis genes induced by B. cinerea over time (temporal) while the second investigated spatial expression of Arabidopsis genes from the point of inoculation. A number of genes were up- and down-regulated specifically at 12 hrs, others at 24 hrs while others were up- and down-regulated at both time points. Similarly, some genes were specifically induced very close to the lesion while others in more distal tissue

Metatranscriptomic analysis of community structure and metabolism of the rhizosphere microbiome.

Plant-microbe interactions in the rhizosphere, the region of soil influenced by plant roots, are integral to biogeochemical cycling, and maintenance of plant health and productivity. Interactions between model plants and microbes are well understood, but relatively little is about the plant microbiome. Here, comparative metatranscriptomics was used to determine taxonomic compositions and metabolic responses of microbes in soil and the rhizospheres of wheat, oat and pea. Additionally a wild-type oat was compared to a mutant (sad1) deficient in production of antifungal avenacins. Analyses of taxonomic compositions and functions based on rRNA and protein coding genes agreed that rhizosphere microbiomes differed from soil and between plant species. Pea had a stronger effect than wheat and oat, suggesting distinct cereal and legume microbiomes. Proportions of eukaryotic rRNA in the oat and pea rhizospheres were more than fivefold higher than in the wheat rhizosphere or soil. Nematodes and bacterivorous protozoa were enriched in all rhizospheres, while the pea rhizosphere was highly enriched for fungi. Only the eukaryotic community was distinct from wild-type oat in the sad1 mutant, suggesting avenacins have a broader role than protecting from fungal pathogens. The addition of an internal RNA standard allowed quantitative determination of global transcriptional activity in each environment. This was generally higher in the rhizospheres, particularly pea, than in soil. Taxa known to possess metabolic traits potentially important for rhizosphere colonisation, plant growth promotion and pathogenesis were selected by plants. Such traits included cellulose and other plant polymer degradation, nitrogen fixation, hydrogen oxidation, methylotrophy and antibiotic production. These functions were also more highly expressed in rhizospheres than soil. Microbes also induced functions involved in chemotaxis, motility, attachment, pathogenesis, responses to oxidative stress, cycling of nitrogen and sulphur, acquisition of phosphorous, iron and other metals, as well as metabolism of a variety of sugars, aromatics, organic and amino acids, many plant species specific. Profiling microbial communities with metatranscriptomics allowed comparison of relative and quantitative abundance of microbes and their metabolism, from multiple samples, across all domains of life, without PCR bias. This revealed profound differences in the taxonomic composition and metabolic functions of rhizosphere microbiomes between crop plants and soil

Synthetic bacterial communities for plant growth promotion

PhD ThesisIncreasing food demands have driven the adoption of new global strategies to intensify productivity without relying on heavy chemical treatments. In the last decades, plant-growth promoting rhizobacteria (PGPR) have emerged as potential biofertilisers and biopesticides in agriculture. The overall aim of this study was to research and develop approaches to genetically engineer PGPR to improve their beneficial activities toward the plant partner. A simplified PGPR community, a Bacillus consortium of three strains, was adopted to study the complexity of the interactions occurring within the consortium and the plant microbiome. Firstly, the comparative genomic analysis of the consortium highlighted the unique and shared features responsible for plant promotion, microbial interaction and cooperation among the strains (niche partitioning, organisation in biofilms with cooperative mechanisms of quorum sensing, cell density control and antibiotic detoxification). Flux balance analysis identified cross-feeding interactions among the strains and the metabolic capability of the consortium to provide nitrogen to the plant, transforming it into forms available for plant utilisation. The consortium PGP potential was then investigated in vitro (LEAP mesocosm assay) and in vivo (pot experiment) on the vegetable crop Brassica rapa. These tests show increased plant growth when the strains were inoculated together rather than individually and when the consortium was used as a supplement of the natural bulk soil microbiome. The in silico study and the plant experiments highlighted areas for genetic improvement of the consortium genomes. Lastly, this work describes the development of a conjugation system that could be used to efficiently engineer non-domesticated bacteria and bacterial communities, such as rhizobacteria and plant microbiomes. The system, based on the plasmid pLS20, was developed in Bacillus subtilis 168 and successfully tested on twenty-three wild type Bacillus strains and three rhizobacillus communities. The research presented here provides tools and approaches for the genetic manipulation of rhizobacterial communities, with the ultimate aim of generating sustainable agricultural bioformulations and sheds light on the complex interactions that can occur in a model microbial PGPR consortia

Dissecting the molecular responses of Sorghum bicolor to Macrophomina phaseolina infection

Doctor of PhilosophyDepartment of Plant PathologyChristopher R. LittleCharcoal rot, caused by the necrotrophic fungus, Macrophomina phaseolina (Tassi) Goid., is an important disease in sorghum (Sorghum bicolor (L.) Moench). The molecular interactions between sorghum and M. phaseolina are poorly understood. In this study, a large-scale RNA-Seq experiment and four follow-up functional experiments were conducted to understand the molecular basis of charcoal rot resistance and/or susceptibility in sorghum. In the first experiment, stalk mRNA was extracted from charcoal-rot-resistant (SC599) and susceptible (Tx7000) genotypes and subjected to RNA sequencing. Upon M. phaseolina inoculation, 8560 genes were differentially expressed between the two genotypes, out of which 2053 were components of 200 known metabolic pathways. Many of these pathways were significantly up-regulated in the susceptible genotype and are thought to contribute to enhanced pathogen nutrition and virulence, impeded host basal immunity, and reactive oxygen (ROS) and nitrogen species (RNS)-mediated host cell death. The paradoxical hormonal regulation observed in pathogen-inoculated Tx7000 was characterized by strongly upregulated salicylic acid and down-regulated jasmonic acid pathways. These findings provided useful insights into induced host susceptibility in response to this necrotrophic fungus at the whole-genome scale. The second experiment was conducted to investigate the dynamics of host oxidative stress under pathogen infection. Results showed M. phaseolina’s ability to significantly increase the ROS and RNS content of two charcoal-rot-susceptible genotypes, Tx7000 and BTx3042. Over-accumulation of nitric oxide (NO) in stalk tissues in the pathogen-inoculated susceptible genotypes was confirmed using a NO-specific fluorescent probe and confocal microscopy. Significantly increased malondialdehyde content confirmed the enhanced oxidative stress experienced by the susceptible genotypes after pathogen inoculation. These findings suggested the contribution of oxidative stress-associated induced cell death on charcoal rot susceptibility under infection. In the third functional experiment, the behavior of the sorghum antioxidant system after pathogen inoculation was investigated. M. phaseolina significantly increased the glutathione s-transferase (GST), glutathione peroxidase (GPX), glutathione reductase (GR), and peroxidase activities of the susceptible genotypes (Tx7000, BTx3042) but not in the resistant genotypes (SC599, SC35). Increased activities of these enzymes in susceptible genotypes may contribute to reduced oxidative stress thus lowering charcoal rot susceptibility. The fourth functional experiment was designed to quantify induced host-derived cell wall degrading enzymes (CWDEs) using crude enzyme mixtures from stalks. A gel diffusion assay revealed significantly increased pectinesterase activity in pathogen-inoculated Tx7000 and BTx3042 while significantly increased polygalacturonase activity was determined by absorbance. Fluorimetric determination of cell extracts revealed significantly increased cellulose degrading enzyme activity in M. phaseolina-inoculated Tx7000 and BTx3042. These findings revealed the pathogen’s ability to promote charcoal rot susceptibility in grain sorghum through induced host CWDEs. The last functional study was designed to profile the stalk tissue lipidome of Tx7000 and SC599 after M. phaseolina inoculation using automated direct infusion electrospray ionization-triple quadrupole mass spectrometry (ESI-MS/MS). M. phaseolina significantly decreased the phytosterol, phosphatidylserine, and ox-lipid contents in Tx7000 while significantly increasing stigmasterol:sitosterol ratio. Except for ox-lipid content, none of the above was significantly affected in resistant SC599. Results suggested the lethal impacts of M. phaseolina inoculation on plastid- and cell- membrane integrity and the lipid-based signaling capacity of Tx7000. Findings shed light on the host lipid classes that contribute to induced charcoal rot susceptibility in grain sorghum