Viable protoplast isolation, organelle visualization and transformation of the globally distributed plant pathogen Phytophthora cinnamomi.

Phytophthora cinnamomi is an oomycete plant pathogen with a host range of almost 5000 plant species worldwide and therefore poses a serious threat to biodiversity. Omics technology has provided significant progress in our understanding of oomycete biology, however, transformation studies of Phytophthora for gene functionalisation are still in their infancy. Only a limited number of Phytophthora species have been successfully transformed and gene edited to elucidate the role of particular genes. There is a need to escalate our efforts to understand molecular processes, gene regulation and infection mechanisms of the pathogen to enable us to develop new disease management strategies. The primary obstacle hindering the advancement of transformation studies in Phytophthora is their challenging and unique nature, coupled with our limited comprehension of why they remain such an intractable system to work with. In this study, we have identified some of the key factors associated with the recalcitrant nature of P. cinnamomi. We have incorporated fluorescence microscopy and flow cytometry along with the organelle-specific dyes, fluorescein diacetate, Hoechst 33342 and MitoTracker™ Red CMXRos, to assess P. cinnamomi-derived protoplast populations. This approach has also provided valuable insights into the broader cell biology of Phytophthora. Furthermore, we have optimized the crucial steps that allow transformation of P. cinnamomi and have generated transformed isolates that express a cyan fluorescent protein, with a transformation efficiency of 19.5%. We therefore provide a platform for these methodologies to be applied for the transformation of other Phytophthora species and pave the way for future gene functionalisation studies.

Chloroplast transformation in new cultivars of tomato through particle bombardment.

A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum L.) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03954-3.

Analysis of plant cuticles and their interactions with agrochemical surfactants using a 3D printed diffusion chamber.

BACKGROUND: Decades of research is available on their effects of single component surfactant on active ingredient diffusion across plant cuticular membranes, but ingredient diffusion is rarely analysed in the presence of commercial surfactants. Also, diffusion studies require expensive or specialized apparatus the  fabrication of which often requires skilled labour and specialized facilities. In this research we have addressed both problems where the effects of four commercially available surfactants on a known tracer molecule were investigated using a 3D printed customized diffusion chamber. RESULTS: As a proof-of-concept a customized 3D printed diffusion chamber was devised using two different thermoplastics and was successfully used in a range of diffusion tests . The effect of various solvents and surfactants on S. lycopersicum cuticular membrane indicated an increased rate of flux of tracer molecules across the membranes. This research has validated the application of 3D printing in diffusion sciences and demonstrated the flexibility and potential of this technique. CONCLUSIONS: Using a 3D printed diffusion apparatus, the effect of commercial surfactants on molecular diffusion through isolated plant membranes was studied. Further, we have included  here the steps involved in material selection, design, fabrication, and post processing procedures for successful recreation of the chamber. The customizability and rapid production process of the 3D printing demonstrates the power of additive manufacturing in the design and use of customizable labware.

Carotenoid Pathway Engineering in Tobacco Chloroplast Using a Synthetic Operon.

The carotenoid pathway in plants has been altered through metabolic engineering to enhance their nutritional value and generate keto-carotenoids, which are widely sought after in the food, feed, and human health industries. In this study, the aim was to produce keto-carotenoids by manipulating the native carotenoid pathway in tobacco plants through chloroplast engineering. Transplastomic tobacco plants were generated that express a synthetic multigene operon composed of three heterologous genes, with Intercistronic Expression Elements (IEEs) for effective mRNA splicing. The metabolic changes observed in the transplastomic plants showed a significant shift towards the xanthophyll cycle, with only a minor production of keto-lutein. The use of a ketolase gene in combination with the lycopene cyclase and hydroxylase genes was a novel approach and demonstrated a successful redirection of the carotenoid pathway towards the xanthophyll cycle and the production of keto-lutein. This study presents a scalable molecular genetic platform for the development of novel keto-carotenoids in tobacco using the Design-Build-Test-Learn (DBTL) approach. This study corroborates chloroplast metabolic engineering using a synthetic biology approach for producing novel metabolites belonging to carotenoid class in industrially important tobacco plant. The synthetic multigene construct resulted in producing a novel metabolite, keto-lutein with high accumulation of xanthophyll metabolites. This figure was drawn using BioRender ( https://www.biorender.com ).

Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops.

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloroplast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogenous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuticals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet.

Chitosan nanoparticles and their combination with methyl jasmonate for the elicitation of phenolics and flavonoids in plant cell suspension cultures.

Productivity enhancement approaches, such as elicitation can overcome the limitations of low metabolite(s) yield in in vitro plant cell culture platforms. Application of biotic/abiotic elicitors triggers molecular responses that lead to a concomitant enhancement in the production of metabolites. Nanoparticles have been tested as alternatives to commonly studied biotic/abiotic elicitors. However, most nanoparticles explored are of metallic origin, which raises concerns about their cytotoxicity, disposal post-elicitation, and may limit downstream applications of metabolites. Here, we report the synthesis and application of biopolymeric methyl jasmonate-loaded chitosan nanoparticles (MJ-CNPs) and empty CNPs (size <100 nm) as nano-elicitors, which were simple to synthesize, cost-effective and safe. Enzymatic and metabolic investigations revealed that MJ-CNPs and empty CNPs improve and prolong phenylalanine ammonia-lyase enzyme activity and production of phenolics and flavonoids. The data provides the first evidence of MJ-CNPs and empty CNPs as nano-elicitors that prolong the production of metabolites in plant cell suspension cultures.

Reduced Genotoxicity of Gold Nanoparticles With Protein Corona in Allium cepa.

Increased usage of gold nanoparticles (AuNPs) in biomedicine, biosensing, diagnostics and cosmetics has undoubtedly facilitated accidental and unintentional release of AuNPs into specific microenvironments. This is raising serious questions concerning adverse effects of AuNPs on off-target cells, tissues and/or organisms. Applications utilizing AuNPs will typically expose the nanoparticles to biological fluids such as cell serum and/or culture media, resulting in the formation of protein corona (PC) on the AuNPs. Evidence for PC altering the toxicological signatures of AuNPs is well studied in animal systems. In this report, we observed significant genotoxicity in Allium cepa root meristematic cells (an off-target bioindicator) treated with high concentrations (≥100 µg/ml) of green-synthesized vanillin capped gold nanoparticles (VAuNPs). In contrast, protein-coated VAuNPs (PC-VAuNPs) of similar concentrations had negligible genotoxic effects. This could be attributed to the change in physicochemical characteristics due to surface functionalization of proteins on VAuNPs and/or differential bioaccumulation of gold ions in root cells. High elemental gold accumulation was evident from µ-XRF mapping in VAuNPs-treated roots compared to treatment with PC-VAuNPs. These data infer that the toxicological signatures of AuNPs are influenced by the biological route that they follow to reach off-target organisms such as plants. Hence, the current findings highlight the genotoxic risk associated with AuNPs, which, due to the enhanced utility, are emerging as new pollutants. As conflicting observations on the toxicity of green-synthesized AuNPs are increasingly reported, we recommend that detailed studies are required to investigate the changes in the toxicological signatures of AuNPs, particularly before and after their interaction with biological media and systems.

Metabolic Engineering of Rice Cells with Vanillin Synthase Gene (VpVAN) to Produce Vanillin.

Vanillin production by metabolic engineering of proprietary microbial strains has gained impetus due to increasing consumer demand for naturally derived products. Here, we demonstrate the use of rice cell cultures metabolically engineered with vanillin synthase gene (VpVAN) as a plant-based alternative to microbial vanillin production systems. VpVAN catalyzes the signature step to convert ferulic acid into vanillin in Vanilla planifolia. As ferulic acid is a phenylpropanoid pathway intermediate in plant cells, rice calli cells are ideal platform for in vivo vanillin synthesis due to the availability of its precursor. In this study, rice calli derived from embryonic rice cells were metabolically engineered with a codon-optimized VpVAN gene using Agrobacterium-mediated transformation. The putative transformants were selected based on their proliferation on herbicide-supplemented N6D medium. Expression of the transgenes were confirmed through a ß-glucuronidase (GUS) reporter assay and polymerase chain reaction (PCR) analysis provided evidence of genetic transformation. The semiquantitative RT-PCR and real-time (RT)-qPCR revealed expression of VpVAN in six transgenic calli lines. High-performance liquid chromatography identified the biosynthesis of vanillin in transgenic calli lines, with the highest yielding line producing 544.72 (± 102.50) µg of vanillin-g fresh calli. This work serves as a proof-of-concept to produce vanillin using metabolically engineered rice cell cultures.

Designer nanoparticles for plant cell culture systems: Mechanisms of elicitation and harnessing of specialized metabolites.

Plant cell culture systems have become an attractive and sustainable approach to produce high-value and commercially significant metabolites under controlled conditions. Strategies involving elicitor supplementation into plant cell culture media are employed to mimic natural conditions for increasing the metabolite yield. Studies on nanoparticles (NPs) that have investigated elicitation of specialized metabolism have shown the potential of NPs to be a substitute for biotic elicitors such as phytohormones and microbial extracts. Customizable physicochemical characteristics allow the design of monodispersed-, stimulus-responsive-, and hormone-carrying-NPs of precise geometries to enhance their elicitation capabilities based on target metabolite/plant cell culture type. We contextualize advances in NP-mediated elicitation, especially stimulation of specialized metabolic pathways, the underlying mechanisms, impacts on gene regulation, and NP-associated cytotoxicity. The novelty of the concept lies in unleashing the potential of designer NPs to enhance yield, harness metabolites, and transform nanoelicitation from exploratory investigations to a commercially viable strategy.

How to Unravel the Key Functions of Cryptic Oomycete Elicitin Proteins and Their Role in Plant Disease.

Pathogens and plants are in a constant battle with one another, the result of which is either the restriction of pathogen growth via constitutive or induced plant defense responses or the pathogen colonization of plant cells and tissues that cause disease. Elicitins are a group of highly conserved proteins produced by certain oomycete species, and their sterol binding ability is recognized as an important feature in sterol-auxotrophic oomycetes. Elicitins also orchestrate other aspects of the interactions of oomycetes with their plant hosts. The function of elicitins as avirulence or virulence factors is controversial and is dependent on the host species, and despite several decades of research, the function of these proteins remains elusive. We summarize here our current understanding of elicitins as either defense-promoting or defense-suppressing agents and propose that more recent approaches such as the use of 'omics' and gene editing can be used to unravel the role of elicitins in host-pathogen interactions. A better understanding of the role of elicitins is required and deciphering their role in host-pathogen interactions will expand the strategies that can be adopted to improve disease resistance and reduce crop losses.

Stimulation of photosynthesis and enhancement of growth and yield in Arabidopsis thaliana treated with amine-functionalized mesoporous silica nanoparticles.

Mesoporous silica nanoparticles (MSNs) of 50 nm diameter particle size with a pore size of approximately 14.7 nm were functionalized with amino groups (Am-MSNs) and the effects of exposure to these positively charged Am-MSNs on each of the life cycle stages of Arabidopsis thaliana were investigated. After growth in half strength MS medium amended with Am-MSNs (0-100 µg/mL) for 7 and 14 days, seed germination rate and seedling growth were significantly increased compared with untreated controls. The seedlings were then transferred to soil and irrigated with Am-MSNs solutions every 3 days until seed harvesting. After four weeks growth in soil, Am-MSNs treated plants showed up-regulation of chlorophyll and carotenoid synthesis-related genes, an increase in the content of photosynthetic pigments and an amplification of plant photosynthetic capacity. All these changes in plants were closely correlated with greater vegetative growth and higher seed yield. In all the experiments, 20 and 50 µg/mL of Am-MSNs were found to be more effective with respect to other treatments, while Am-MSNs at the highest level of 100 µg/mL did not result in oxidative stress or cell membrane damage in the exposed plants. To the best of our knowledge, this is the first report evaluating both physiological and molecular responses following exposure to plants of these specific Am-MSNs throughout their whole life cycle. Overall, these findings indicate that following exposure Am-MSNs play a major role in the increase in seed germination, biomass, photosynthetic pigments, photosynthetic capacity and seed yield in A. thaliana.

Next-generation metabolic engineering approaches towards development of plant cell suspension cultures as specialized metabolite producing biofactories.

Plant cell suspension culture (PCSC) has emerged as a viable technology to produce plant specialized metabolites (PSM). While Taxol® and ginsenoside are two examples of successfully commercialized PCSC-derived PSM, widespread utilization of the PCSC platform has yet to be realized primarily due to a lack of understanding of the molecular genetics of PSM biosynthesis. Recent advances in computational, molecular and synthetic biology tools provide the opportunity to rapidly characterize and harness the specialized metabolic potential of plants. Here, we discuss the prospects of integrating computational modeling, artificial intelligence, and precision genome editing (CRISPR/Cas and its variants) toolboxes to discover the genetic regulators of PSM. We also explore how synthetic biology can be applied to develop metabolically optimized PSM-producing native and heterologous PCSC systems. Taken together, this review provides an interdisciplinary approach to realize and link the potential of next-generation computational and molecular tools to convert PCSC into commercially viable PSM-producing biofactories.

Nanoapplication of a Resistance Inducer to Reduce Phytophthora Disease in Pineapple (Ananas comosus L.).

Treatment of plants with a variety of abiotic and biotic inducers causes induced resistance to pathogen attack. In this study, the effect of four resistance inducers on plant diseases caused by Phytophthora cinnamomi was screened in vivo initially by using lupin, a susceptible model plant. Lupin pretreated with 0.5 mM salicylic acid (SA) showed effective resistance against P. cinnamomi with restricted lesions. Then, mesoporous silica nanoparticles (MSNs) with particle size around 20 nm and approximate pore size of 3.0 nm were synthesized and functionalized for loading and importing SA to pineapple plantlets. Decanethiol gatekeepers were introduced to the surface of MSNs via glutathione (GSH)-cleavable disulfide linkages to cover the pore entrance, which was confirmed through using Raman spectroscopy. Through free diffusion, the loading efficiency of SA in MSNs gated with gatekeepers was 11.7%, but was lower in MSNs without gatekeepers (8.0%). In addition, in vitro release profile of SA from gatekeeper-capped MSNs indicated that higher concentrations of GSH resulted in more cargo release. Moreover, the experiments in planta showed that the application of MSNs as a resistance inducer delivery system significantly improved pineapple resistance to P. cinnamomi in terms of inhibiting lesion development and improving root growth of infected plants, compared to the use of free SA and MSNs without gatekeepers. The analysis of SA, GSH, and defense-related genes, of PR1 and PR5, further confirmed that the slow and prolonged release of SA from MSNs inside the roots of pineapple plants was achieved through a redox-stimuli release mechanism. Therefore, the application of MSNs with redox-responsive gatekeepers has shown great potential as an efficient tool for delivering chemicals into plants in a controllable way.

Vanillin mediated green synthesis and application of gold nanoparticles for reversal of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates.

Antimicrobial resistance (AMR) is a serious concern in pathogenic bacteria. As a new approach to addressing AMR, we report here the green synthesis of vanillin capped gold nanoparticles (VAuNPs) using the popular flavouring molecule vanillin (C8H8O3) as a reducing and capping agent. Physicochemical characterization revealed that the synthesised VAuNPs were stable and crystalline in nature. VAuNPs were non-bactericidal even at high concentration (>2000 µg/ml). The antibiotic potentiation activity was studied in combination with seven widely used antibiotics against extremely drug resistant (XDR) Pseudomonas aeruginosa. Major reductions in minimum inhibitory concentrations (MIC, 10-14-folds) of the antibiotics meropenem (10 fold) and trimethoprim (14 fold) were observed in the presence of VAuNPs (50 µg/ml). Furthermore, it was found that VAuNPs in combination with meropenem or trimethoprim provided 1.5-3-fold better potentiation effects than that of vanillin alone. Use of an ethidium bromide agar cart wheel assay indicated that VAuNPs can block the activity of efflux pumps. High reduction in the MIC of antibiotics was therefore attributed to the efflux pump repression activity of VAuNPs. Further, RT-qPCR of clinically relevant MexAB-OprM efflux pump components showed down-regulation in mexB and OprM transcripts in VAuNPs treated P. aeruginosa clinical isolates. Our results reveal that VAuNPs impart susceptibility to the last line antibiotics meropenem, trimethoprim and few widely used antibiotics in XDR P. aeruginosa clinical isolates that display resistance to these antibiotics. Therefore, this study indicate the ability of VAuNPs and vanillin to be used as antibiotic adjuvants for inhibiting bacterial efflux pumps to potentiate antibiotics for addressing AMR problem affecting human health and environment.

Enhanced polycyclic aromatic hydrocarbons degradation in rhizosphere soil planted with tall fescue: Bacterial community and functional gene expression mechanisms.

To investigate the bacterial mechanisms of polycyclic aromatic hydrocarbons (PAHs) degradation in an aged-contaminated agricultural soil planted with tall fescue (Festuca arundinacea), a rhizo-box experiment was carried out for 60 d. Shifts in bacterial community structure in the soils during the experiment were performed using denaturing gradient gel electrophoresis. The abundance and activity of total bacteria and PAH-degraders were measured by quantification of 16S rDNA, PAH-ring hydroxylating dioxygenase (PAH-RHDα) genes and their transcripts, respectively. The residual PAH concentrations were monitored using high-performance liquid chromatography analysis. Results showed that the removal percentage of total PAHs in rhizosphere soil was 11% higher than that in unplanted soil. Soil bacteria were dominated by Alphaproteobacteria (48.4%) and Gammaproteobacteria (25.8%). Tall fescue positively affected the abundance and activity of total bacteria in the soil, and stimulated RHDα gram-negative (GN) gene expression while inhibiting RHDα gram-positive gene expression. PAH dissipation in rhizosphere soil could be ascribed to modifications in the bacterial community structure, increase in the abundance of PAH-degraders, and enhancement of the RHDα GN gene expression during the incubation.

The influence of ascorbic acid on root growth and the root apical meristem in Arabidopsis thaliana.

Cell division is a fundamental biological process governed by molecular networks that are initiated in the apical meristems of plants. l-ascorbic acid (AsA) commonly known as vitamin C is a crucial molecular modulator involved in cell proliferation. In this study, we used AsA application to Arabidopsis and four AsA pathway mutants to investigate the influence of AsA on the root apical meristem (RAM) and root growth. Treatment of seeds of wild-type Col-0 with AsA prior to sowing showed a significant increase in the activity of cell division of the RAM, root growth rate and root length when compared with untreated seeds. Seedlings of the AsA pathway mutant vtc1-1 showed a significant reduction in the level of AsA and a significant increase in the number of quiescent cells in the RAM when compared with Col-0. Cell proliferation was reduced in the AsA pathway mutants vtc1-1, dhar1, vtc5-1, apx1, respectively, however, root growth decreased significantly only in vtc1-1 when compared with Col-0. In addition, hydrogen peroxide (H2O2) levels were shown to increase in the AsA pathway mutants, with the highest level of H2O2 found in vtc1-1. AsA is also shown to have an indirect influence in inducing periclinal division as a reduced level was found in vtc1-1. Therefore, in this study, we found that AsA had an influence on cell proliferation and root growth and VTC1 was shown to be a key modulator of H2O2 levels. These findings open the door for further studies to reveal the involvement of AsA in cell proliferation and the interaction between AsA and H2O2 on cell polarity in the RAM and potentially the shoot apical meristem.

Delivery of Abscisic Acid to Plants Using Glutathione Responsive Mesoporous Silica Nanoparticles.

An intracellular glutathione (GSH) responsive phytochemical delivery system based on thiol gated mesoporous silica nanoparticles (MSNs) was developed and tested on the model plant Arabidopsis thaliana. In the present study, monodispersed MSNs with particle diameters of ~20 nm and pore sizes of ~2.87 nm were synthesized and modified. Abscisic acid (ABA), a key phytohormone, was entrapped in the mesopores of MSNs and then the pore entrances of MSNs were covered with decanethiol gatekeepers through GSH-cleavable disulfide linkages. An in vitro release test of ABA from decanethiol gated MSNs proved that there was efficient loading and entrapment of phytochemicals in the absence of a GSH redox trigger. Most importantly, in planta experiments demonstrated that GSH-mediated release of ABA from the pores of MSNs significantly reduced the leaf stomatal aperture and inhibited water loss of treated plants. Moreover, compared with the usage of free ABA, the controlled release of the encapsulated phytohormone from MSNs markedly prolonged the expression of the ABA inducible marker gene (AtGALK2) and finally, improved the drought resistance ability of Arabidopsis seedlings under drought stress. Therefore, the concept of using short-chain molecules as gatekeepers to encapsulate biomolecules in MSNs was demonstrated. The application of MSNs with redox-responsive gatekeepers has been shown in this study to be a potential and efficient technique to deliver phytochemicals into plants and release them in a controllable fashion.

Polycyclic aromatic hydrocarbons bioavailability in industrial and agricultural soils: Linking SPME and Tenax extraction with bioassays.

The aims of this study were to evaluate the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in industrial and agricultural soils using chemical methods and a bioassay, and to study the relationships between the methods. This was conducted by comparing the quantities of PAHs extracted from two manufactured gas plant (MGP) soils and an agricultural soil with low level contamination by solid-phase micro-extraction (SPME) and Tenax-TA extraction with the quantities taken up by the earthworm (Eisenia fetida). In addition, a biodegradation experiment was conducted on one MGP soil (MGP-A) to clarify the relationship between PAH removal by biodegradation and the variation in PAH concentrations in soil pore water. Results demonstrated that the earthworm bioassay could not be used to examine PAH bioavailability in the tested MGP soils; which was the case even in the diluted MGP-A soils after biodegradation. However, the bioassay was successfully applied to the agricultural soil. These results suggest that earthworms can only be used for bioassays in soils with low toxicity. In general, rapidly desorbing concentrations extracted by Tenax-TA could predict PAH concentrations accumulated in earthworms (R2=0.66), while SPME underestimated earthworm concentrations by a factor of 2.5. Both SPME and Tenax extraction can provide a useful tool to predict PAH bioavailability for earthworms, but Tenax-TA extraction was proven to be a more sensitive and precise method than SPME for the prediction of earthworm exposure in the agricultural soil.

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

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.

Active defence by an Australian native host, Lomandra longifolia, provides resistance against Phytophthora cinnamomi.

Resistance is rare against the oomycete plant pathogen Phytophthora cinnamomi Rands. Only a limited number of species have been recorded as field-resistant species in Australia. However, understanding the nature of resistance of those species when grown under controlled conditions is challenging because of their slow growth and the inherent difficulties of working with a root pathogen. We assessed the Australian native species, Lomandra longifolia Labill., as a resistant species by analysing in detail the response of roots to infection by P. cinnamomi in a series of comparative tests with Lupinus angustifolius L., a highly susceptible species. Following inoculation of L. longifolia roots, lesion length and colonisation percentage were significantly less than in roots of the susceptible species. Moreover, there was no statistical difference in root growth rate, whole-plant FW and leaf relative chlorophyll content between controls and inoculated L. longifolia. We then examined three key cellular responses that are related to resistance: the production of the reactive oxygen species, H2O2, callose formation and lignin deposition in L. longifolia roots following inoculation with P. cinnamomi. The upregulation of these resistance-related components in the early hours after inoculation suggested their involvement in resistance and that this is controlled by the coordinated response of multiple components. Resistance assessment and a detailed investigation of cellular resistance components along with gene expression analysis provides a platform for further understanding of the mechanisms of resistance against this generalist pathogen and presents opportunities for manipulating susceptible species for disease resistance.