James Rookes

Heterotrimeric G Proteins Facilitate Arabidopsis Resistance to Necrotrophic Pathogens and Are Involved in Jasmonate Signaling

Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the Gβγ dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional Gα or Gβ subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in Gβ-deficient mutants while Gα-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in Gβ-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, Gβ-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate-induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the Gα-deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gβγ functional subunit but not by Gα. We hypothesize that Gβγ acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.

Phytophthora cinnamomi and Australia's biodiversity : impacts, predictions and progress towards control

Phytophthora cinnamomi continues to cause devastating disease in Australian native vegetation and consequently the disease is listed by the Federal Government as a process that is threatening Australia’s biodiversity. Although several advances have been made in our understanding of how this soil-borne pathogen interacts with plants and of how we may tackle it in natural systems, our ability to control the disease is limited. The pathogen occurs widely across Australia but the severity of its impact is most evident within ecological communities of the south-west and south-east of the country. A regional impact summary for all states and territories shows the pathogen to be the cause of serious disease in numerous species, a significant number of which are rare and threatened. Many genera of endemic taxa have a high proportion of susceptible species including the iconic genera Banksia, Epacris and Xanthorrhoea. Long-term studies in Victoria have shown limited but probably unsustainable recovery of susceptible vegetation, given current management practices. Management of the disease in conservation reserves is reliant on hygiene, the use of chemicals and restriction of access, and has had only limited effectiveness and not provided complete control. The deleterious impacts of the disease on faunal habitat are reasonably well documented and demonstrate loss of individual animal species and changes in population structure and species abundance. Few plant species are known to be resistant to P. cinnamomi; however, investigations over several years have discovered the mechanisms by which some plants are able to survive infection, including the activation of defence-related genes and signalling pathways, the reinforcement of cell walls and accumulation of toxic metabolites. Manipulation of resistance and resistance-related mechanisms may provide avenues for protection against disease in otherwise susceptible species. Despite the advances made in Phytophthora research in Australia during the past 40 years, there is still much to be done to give land managers the resources to combat this disease. Recent State and Federal initiatives offer the prospect of a growing and broader awareness of the disease and its associated impacts. However, awareness must be translated into action as time is running out for the large number of susceptible, and potentially susceptible, species within vulnerable Australian ecological communities.

Heterotrimeric G Protein γ Subunits Provide Functional Selectivity in Gβγ Dimer Signaling in Arabidopsis

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Gα and Gβ subunit genes and two Gγ subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Gγ genes revealed specialized roles for each Gγ subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gβγ1 and Gβγ2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Gγ subunit in this root response differs, with Gβγ1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gβγ2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gβγ signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Gγ subunits form functional Gβγ dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein–mediated signaling in plants.

Heterotrimeric G proteins‐mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid‐, jasmonic acid/ethylene‐ and abscisic acid‐mediated defense signaling

Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling.

Analysis of global host gene expression during the primary phase of the Arabidopsis thaliana–Plasmodiophora brassicae interaction

Microarray analysis was used to investigate changes in host gene expression during the primary stages of the interaction between the susceptible plant Arabidopsis thaliana (L.) Heynh ecotype Col-0 and the biotrophic pathogen Plasmodiophora brassicae Woronin. Analyses were conducted at 4, 7 and 10 days after inoculation (DAI) and revealed significant induction or suppression of a relatively low number of genes in a range of functional categories. At 4 DAI, there was induced expression of several genes known to be critical for pathogen recognition and signal transduction in other resistant host-pathogen interactions. As the pathogen further colonised root tissue and progressed through the primary plasmodium stage to production of zoosporangia at 7 and 10 DAI, respectively, fewer genes showed changes in expression. The microarray results were validated by examining a subset of induced genes at 4 DAI by quantitative real-time reverse transcriptase PCR (RT-qPCR) analysis all of which correlated positively with the microarray data. The two A. thaliana mutants jar1 and coiI tested were found to be susceptible to P. brassicae. The involvement of defence-related hormones in the interaction was further investigated and the findings indicate that addition of salicylic acid can suppress clubroot disease in A. thaliana plants.

Functionalized Mesoporous Silica Nanoparticles with Redox-Responsive Short-Chain Gatekeepers for Agrochemical Delivery

The controlled release of salicylic acid (SA), a key phytohormone, was mediated by using a novel decanethiol gatekeeper system grafted onto mesoporous silica nanoparticles (MSNs). The decanethiol was conjugated only to the external surfaces of the MSNs through glutathione (GSH)-cleavable disulfide linkages and the introduction of a process to assemble gatekeepers only on the outer surface so that the mesopore area can be maintained for high cargo loading. Raman and nitrogen sorption isotherm analyses confirmed the successful linkage of decanethiol to the surface of MSNs. The in vitro release of SA from decanethiol gated MSNs indicated that the release rate of SA in an environment with a certain amount of GSH was significantly higher than that without GSH. More importantly, in planta experiments showed the release of SA from decanethiol gated MSNs by GSH induced sustained expression of the plant defense gene PR-1 up to 7 days after introduction, while free SA caused an early peak in PR-1 expression which steadily decreased after 3 days. This study demonstrates the redox-responsive release of a phytohormone in vitro and also indicates the potential use of MSNs in planta as a controlled agrochemical delivery system.

Transcriptional profiling of Zea mays roots reveals roles for jasmonic acid and terpenoids in resistance against Phytophthora cinnamomi

Phytophthora cinnamomi is a soil-borne plant pathogen that has caused widespread damage to vulnerable native ecosystems and agriculture systems across the world and shows no sign of abating. Management of the pathogen in the natural environment is difficult and the options are limited. In order to discover more about how resistant plants are able to defend themselves against this generalist pathogen, a microarray study of plant gene expression following root inoculation with P. cinnamomi was undertaken. Zea mays was used as a resistant model plant, and microarray analysis was conducted using the Affymetrix GeneChip Maize Genome Array on root samples collected at 6- and 24-h post-inoculation. Over 300 genes were differentially expressed in inoculated roots compared with controls across the two time points. Following Gene Ontology enrichment analysis and REVIGO visualisation of the up-regulated genes, many were implicated in plant defence responses to biotic stress. Genes that were up-regulated included those involved in phytoalexin biosynthesis and jasmonic acid/ethylene biosynthesis and other defence-related genes including those encoding glutathione S-transferases and serine-protease inhibitors. Of particular interest was the identification of the two most highly up-regulated genes, terpene synthase11 (Tps11) and kaurene synthase2 (An2), which are both involved in production of terpenoid phytoalexins. This is the first study that has investigated gene expression at a global level in roots in response to P. cinnamomi in a model plant species and provides valuable insights into the mechanisms involved in defence.

Transcriptomics-based analysis using RNA-Seq of the coconut (Cocos nucifera) leaf in response to yellow decline phytoplasma infection.

Abstract Invasive phytoplasmas wreak havoc on coconut palms worldwide, leading to high loss of income, food insecurity and extreme poverty of farmers in producing countries. Phytoplasmas as strictly biotrophic insect-transmitted bacterial pathogens instigate distinct changes in developmental processes and defence responses of the infected plants and manipulate plants to their own advantage; however, little is known about the cellular and molecular mechanisms underlying host–phytoplasma interactions. Further, phytoplasma-mediated transcriptional alterations in coconut palm genes have not yet been identified. This study evaluated the whole transcriptome profiles of naturally infected leaves of Cocos nucifera ecotype Malayan Red Dwarf in response to yellow decline phytoplasma from group 16SrXIV, using RNA-Seq technique. Transcriptomics-based analysis reported here identified genes involved in coconut innate immunity. The number of down-regulated genes in response to phytoplasma infection exceeded the number of genes up-regulated. Of the 39,873 differentially expressed unigenes, 21,860 unigenes were suppressed and 18,013 were induced following infection. Comparative analysis revealed that genes associated with defence signalling against biotic stimuli were significantly overexpressed in phytoplasma-infected leaves versus healthy coconut leaves. Genes involving cell rescue and defence, cellular transport, oxidative stress, hormone stimulus and metabolism, photosynthesis reduction, transcription and biosynthesis of secondary metabolites were differentially represented. Our transcriptome analysis unveiled a core set of genes associated with defence of coconut in response to phytoplasma attack, although several novel defence response candidate genes with unknown function have also been identified. This study constitutes valuable sequence resource for uncovering the resistance genes and/or susceptibility genes which can be used as genetic tools in disease resistance breeding.

Nanostructured liquid crystalline particles as an alternative delivery vehicle for plant agrochemicals.

Agrochemical spray formulations applied to plants are often mixed with surfactants that facilitate delivery of the active ingredient. However, surfactants cause phytotoxicity and off-target effects in the environment. We propose the use of nanostructured liquid crystalline particles (NLCP) as an alternative to surfactant-based agrochemical delivery. For this, we have compared the application of commercial surfactants, di (2-ethylhexyl) sulfosuccinate and alkyl dimethyl betaine, with NLCP made from phytantriol, at concentrations of 0.1%, 1% and 5% on the adaxial surface of leaves of four plant species Ttriticum aestivum (wheat), Zea mays (maize), Lupinus angustifolius (lupin), and Arabidopsis thaliana. In comparison with the application of surfactants there was less phytotoxicity on leaves of each species following treatment with NLCP. Following treatment of leaves with NLCP analysis of cuticular wax micromorphology revealed less wax solubilization in the monocot species. The results clearly show that there are advantages in the use of NLCP rather than surfactants for agrochemical delivery.

Plant–pathogen interactions: toward development of next-generation disease-resistant plants

Abstract Briskly evolving phytopathogens are dire threats to our food supplies and threaten global food security. From the recent advances made toward high-throughput sequencing technologies, understanding of pathogenesis and effector biology, and plant innate immunity, translation of these means into new control tools is being introduced to develop durable disease resistance. Effectoromics as a powerful genetic tool for uncovering effector-target genes, both susceptibility genes and executor resistance genes in effector-assisted breeding, open up new avenues to improve resistance. TALENs (Transcription Activator-Like Effector Nucleases), engineered nucleases and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are breakthrough and powerful techniques for genome editing, providing efficient mechanisms for targeted crop protection strategies in disease resistance programs. In this review, major advances in plant disease management to confer durable disease resistance and novel strategies for boosting plant innate immunity are highlighted.