Botrytis cinerea Loss and Restoration of Virulence during In Vitro Culture Follows Flux in Global DNA Methylation.

Pathogenic fungi can lose virulence after protracted periods of culture, but little is known of the underlying mechanisms. Here, we present the first analysis of DNA methylation flux at a single-base resolution for the plant pathogen B. cinerea and identify differentially methylated genes/genomic regions associated with virulence erosion during in vitro culture. Cultures were maintained for eight months, with subcultures and virulence testing every month. Methylation-sensitive amplified polymorphisms were performed at monthly intervals to characterise global changes to the pathogen's genome during culture and also on DNA from mycelium inoculated onto Arabidopsis thaliana after eight months in culture. Characterisation of culture-induced epialleles was assessed by whole-genome re-sequencing and whole-genome bisulfite sequencing. Virulence declined with time in culture and recovered after inoculation on A. thaliana. Variation detected by methylation-sensitive amplified polymorphisms followed virulence changes during culture. Whole-genome (bisulfite) sequencing showed marked changes in global and local methylation during culture but no significant genetic changes. We imply that virulence is a non-essential plastic character that is at least partly modified by the changing levels of DNA methylation during culture. We hypothesise that changing DNA methylation during culture may be responsible for the high virulence/low virulence transition in B. cinerea and speculate that this may offer fresh opportunities to control pathogen virulence.

Nitric oxide donor, sodium nitroprusside modulates hydrogen sulfide metabolism and cysteine homeostasis to aid the alleviation of chromium toxicity in maize seedlings (Zea mays L.).

The current research aimed to assess the protective role of nitric oxide (NO) against chromium (Cr) toxicity in maize seedlings. Chromium (200 µM) lowered osmotic potential in epicotyls and mostly in radicles (by 38% and 63%, respectively) as compared to the control. Sodium nitroprusside (SNP, NO donor) restored seedling biomass (+90% for both organs) and water potential, whereas application of Nω-nitro-L-arginine methylester (L-NAME, a NOS inhibitor) increased sensitivity to Cr. SNP suppressed Cr-triggered proline accumulation by inhibiting Δ1-pyrroline-5-carboxylate synthetase activity and stimulating proline dehydrogenase activity, leading to glutamate over-accumulation (~30% for both organs). Cr stimulated cysteine metabolism and this was further enhanced by SNP which stimulated serine acetyl-transferase and O-acetylserine (thiol) lyase activities. This was followed by an increase in endogenous hydrogen sulfide (H2S) generation by up-regulating L-cysteine desulfhydrase (+205%), D-cysteine desulfhydrase (+150%) and cyanoalanine synthase (+65%) activities in radicles compared to Cr-treatments plants. These positive effects were reduced in L-NAME compared to control. Combined Cr+SNP affected the levels of compounds involved in glutathione metabolism (γ-glutamyl-cysteinyl, γ-glutamyl-cysteinyl-clycine, γ-cysteinyl-glycine, and glycine.). All together, our findings indicate that NO and elicited cellular H2S act synergistically to alleviate Cr stress in maize seedlings by influencing a metabolic interplay between cysteine, proline, and glutathione.

Functions of silicon in plant drought stress responses.

Silicon (Si), the second most abundant element in Earth's crust, exerts beneficial effects on the growth and productivity of a variety of plant species under various environmental conditions. However, the benefits of Si and its importance to plants are controversial due to differences among the species, genotypes, and the environmental conditions. Although Si has been widely reported to alleviate plant drought stress in both the Si-accumulating and nonaccumulating plants, the underlying mechanisms through which Si improves plant water status and maintains water balance remain unclear. The aim of this review is to summarize the morphoanatomical, physiological, biochemical, and molecular processes that are involved in plant water status that are regulated by Si in response to drought stress, especially the integrated modulation of Si-triggered drought stress responses in Si accumulators and intermediate- and excluder-type plants. The key mechanisms influencing the ability of Si to mitigate the effects of drought stress include enhancing water uptake and transport, regulating stomatal behavior and transpirational water loss, accumulating solutes and osmoregulatory substances, and inducing plant defense- associated with signaling events, consequently maintaining whole-plant water balance. This study evaluates the ability of Si to maintain water balance under drought stress conditions and suggests future research that is needed to implement the use of Si in agriculture. Considering the complex relationships between Si and different plant species, genotypes, and the environment, detailed studies are needed to understand the interactions between Si and plant responses under stress conditions.

Non-Targeted Metabolite Profiling Reveals Host Metabolomic Reprogramming during the Interaction of Black Pepper with Phytophthora capsici.

Phytophthora capsici is one of the most destructive pathogens causing quick wilt (foot rot) disease in black pepper (Piper nigrum L.) to which no effective resistance has been defined. To better understand the P. nigrum-P. capsici pathosystem, we employed metabolomic approaches based on flow-infusion electrospray-high-resolution mass spectrometry. Changes in the leaf metabolome were assessed in infected and systemic tissues at 24 and 48 hpi. Principal Component Analysis of the derived data indicated that the infected leaves showed a rapid metabolic response by 24 hpi whereas the systemic leaves took 48 hpi to respond to the infection. The major sources of variations between infected leaf and systemic leaf were identified, and enrichment pathway analysis indicated, major shifts in amino acid, tricarboxylic acid cycle, nucleotide and vitamin B6 metabolism upon infection. Moreover, the individual metabolites involved in defensive phytohormone signalling were identified. RT-qPCR analysis of key salicylate and jasmonate biosynthetic genes indicated a transient reduction of expression at 24 hpi but this increased subsequently. Exogenous application of jasmonate and salicylate reduced P. capsici disease symptoms, but this effect was suppressed with the co-application of abscisic acid. The results are consistent with abscisic acid reprogramming, salicylate and jasmonate defences in infected leaves to facilitate the formation of disease. The augmentation of salicylate and jasmonate defences could represent an approach through which quick wilt disease could be controlled in black pepper.

Transcriptomic Characterization of Nitrate-Enhanced Stevioside Glycoside Synthesis in Stevia (Stevia rebaudiana) Bertoni.

Nitrogen forms (nitrate (NO3-) or ammonium (NH4+)) are vital to plant growth and metabolism. In stevia (Stevia rebaudiana), it is important to assess whether nitrogen forms can influence the synthesis of the high-value terpene metabolites-steviol glycosides (SGs), together with the underlying mechanisms. Field and pot experiments were performed where stevia plants were fertilized with either NO3- or NH4+ nutrition to the same level of nitrogen. Physiological measurements suggested that nitrogen forms had no significant impact on biomass and the total nitrogen content of stevia leaves, but NO3--enhanced leaf SGs contents. Transcriptomic analysis identified 397 genes that were differentially expressed (DEGs) between NO3- and NH4+ treatments. Assessment of the DEGs highlighted the responses in secondary metabolism, particularly in terpenoid metabolism, to nitrogen forms. Further examinations of the expression patterns of SGs synthesis-related genes and potential transcription factors suggested that GGPPS and CPS genes, as well as the WRKY and MYB transcription factors, could be driving N form-regulated SG synthesis. We concluded that NO3-, rather than NH4+, can promote leaf SG synthesis via the NO3--MYB/WRKY-GGPPS/CPS module. Our study suggests that insights into the molecular mechanism of how SG synthesis can be affected by nitrogen forms.

Nitrate mediated resistance against Fusarium infection in cucumber plants acts via photorespiration.

Fusarium wilt is one of the major biotic factors limiting cucumber (Cucumis sativus L.) growth and yield. The outcomes of cucumber-Fusarium interactions can be influenced by the form of nitrogen nutrition (nitrate [NO3- ] or ammonium [NH4+ ]); however, the physiological mechanisms of N-regulated cucumber disease resistance are still largely unclear. Here, we investigated the relationship between nitrogen forms and cucumber resistance to Fusarium infection. Our results showed that on Fusarium infection, NO3- feeding decreased the levels of the fungal toxin, fusaric acid, leaf membrane oxidative, organelle damage and disease-associated loss in photosynthesis. Metabolomic analysis and gas-exchange measurements linked NO3- mediated plant defence with enhanced leaf photorespiration rates. Cucumber plants sprayed with the photorespiration inhibitor isoniazid were more susceptible to Fusarium and there was a negative correlation between photorespiration rate and leaf membrane injury. However, there were positive correlations between photorespiration rate, NO3- assimilation and the tricarboxylic acid (TCA) cycle. This provides a potential electron sink or the peroxisomal H2 O2 catalysed by glycolate oxidase. We suggest that the NO3- nutrition enhanced cucumber resistance against Fusarium infection was associated with photorespiration. Our findings provide a novel insight into a mechanism involving the interaction of photorespiration with nitrogen forms to drive wider defence.

The cross-kingdom roles of mineral nutrient transporters in plant-microbe relations.

The regulation of plant physiology by plant mineral nutrient transporter (MNT) is well understood. Recently, the extensive characterization of beneficial and pathogenic plant-microbe interactions has defined the roles for MNTs in such relationships. In this review, we summarize the roles of diverse nutrient transporters in the symbiotic or pathogenic relationships between plants and microorganisms. In doing so, we highlight how MNTs of plants and microbes can act in a coordinated manner. In symbiotic relationships, MNTs play key roles in the establishment of the interaction between the host plant and rhizobium or mycorrhizae as well in the subsequent coordinated transport of nutrients. Additionally, MNTs may also regulate the colonization or degeneration of symbiotic microorganisms by reflecting the nutrient status of the plant and soil. This allows the host plant obtain nutrients from the soil in the most optimal manner. With pathogenic-interactions, MNTs influence pathogen proliferation, the efficacy of the host's biochemical defense and related signal transduction mechanisms. We classify the MNT effects in plant-pathogen interactions as either indirect by influencing the nutrient status and fitness of the pathogen, or direct by initiating host defense mechanisms. While such observations indicate the fundamental importance of MNTs in governing the interactions with a range of microorganisms, further work is needed to develop an integrative understanding of their functions.

Zinc and Copper Enhance Cucumber Tolerance to Fusaric Acid by Mediating Its Distribution and Toxicity and Modifying the Antioxidant System.

Fusaric acid (FA), the fungal toxin produced by Fusarium oxysporum, plays a predominant role in the virulence and symptom development of Fusarium wilt disease. As mineral nutrients can be protective agents against Fusarium wilt, hydroponic experiments employing zinc (Zn) and copper (Cu) followed by FA treatment were conducted in a glasshouse. FA exhibited strong phytotoxicity on cucumber plants, which was reversed by the addition of Zn or Cu. Thus, Zn or Cu dramatically reduced the wilt index, alleviated the leaf or root cell membrane injury and mitigated against the FA inhibition of plant growth and photosynthesis. Cucumber plants grown with Zn exhibited decreased FA transportation to shoots and a 17% increase in toxicity mitigation and showed minimal hydrogen peroxide, lipid peroxidation level with the increased of antioxidant enzymes activity in both roots and leaves. Cucumber grown with additional Cu absorbed less FA but showed more toxicity mitigation at 20% compared to with additional Zn and exhibited decreased hydrogen peroxide level and increased antioxidant enzymes activity. Thus, adding Zn or Cu can decrease the toxicity of the FA by affecting the absorption or transportation of the FA in plants and mitigate toxicity possibly through chelation. Zn and Cu modify the antioxidant system to scavenge hydrogen peroxide for suppressing FA induction of oxidative damage. Our experiments could provide a theoretical basis for the direct application of micro-fertilizer as protective agents in farming.

Negative effects of the simulated nitrogen deposition on plant phenolic metabolism: A meta-analysis.

Phenolic compounds constitute probably the largest group of plant secondary metabolites and have key roles in plant metabolism. Simulated nitrogen (N) deposition is important to agriculture and has considerable impacts on plant phenolic metabolism but a systematic understanding of such effects is lacking. We here synthesized results from 123 articles and evaluated the responses of plant biomass, in vivo N status, soluble sugar concentrations, carbon (C)/N ratios and multiple phenolic compounds to the simulated N deposition. This meta-analysis showed that the simulated N deposition significantly increased plant biomass and N content but reduced the concentrations of phenolic compounds in a dose-depended manner. This was linked to the suppression of phenolic generating phenylalanine ammonia_lyase activity and key associated gene expression by the simulated N deposition. Total phenolic concentrations were negatively related to biomass but were positively correlated with C/N and soluble sugar contents. Overall, our results indicated adverse effects of simulated N deposition on phenolic metabolism which could compromise key aspects of crop quality and are apparently hidden by positive effects on plant biomass. Our findings have significant ecological and biological implications for plant phenolic metabolism facing global N deposition.

Nitrate Stabilizes the Rhizospheric Fungal Community to Suppress Fusarium Wilt Disease in Cucumber.

Nitrogen forms can regulate soil-borne Fusarium wilt suppression, but the related mechanisms are largely unknown, especially possible action via the rhizospheric microbial community. Soil analysis, MiSeq high-throughput sequencing analysis, community diversity, and network analysis were used to characterize the impact of different nitrogen forms (nitrate and ammonium) on rhizospheric fungal communities and the contribution of nitrate to the suppression to Fusarium oxysporum f. sp. cucumerinum compared with ammonium. Nitrate-grown cucumber showed a lower disease index and F. oxysporum f. sp. cucumerinum abundance in the rhizosphere. In comparisons with ammonium nutrients, nitrate-fed plants maintained a higher soil rhizosphere pH, microbial biomass carbon content, microbial biomass nitrogen content, as well as fungal community richness and diversity following F. oxysporum f. sp. cucumerinum incubation. All these factors were negatively related with disease index. Network analysis showed fewer nodes and edges in the ammonium treatments compared with nitrate treatments. The relative abundance of Pathotroph-Saprotroph, Pathotroph-Saprotroph-Symbiotroph, and Saprotroph fungi explained 82% of the variability of rhizosphere F. oxysporum f. sp. cucumerinum abundance. In conclusion, after pathogen inoculation under nitrate nutrition, the less-affected microbial composition, community diversity, and community internal relations, which resulted from the more diverse and robust microbial population, potentially contributed to greater Fusarium wilt suppression.

Unravelling the Roles of Nitrogen Nutrition in Plant Disease Defences.

Nitrogen (N) is one of the most important elements that has a central impact on plant growth and yield. N is also widely involved in plant stress responses, but its roles in host-pathogen interactions are complex as each affects the other. In this review, we summarize the relationship between N nutrition and plant disease and stress its importance for both host and pathogen. From the perspective of the pathogen, we describe how N can affect the pathogen's infection strategy, whether necrotrophic or biotrophic. N can influence the deployment of virulence factors such as type III secretion systems in bacterial pathogen or contribute nutrients such as gamma-aminobutyric acid to the invader. Considering the host, the association between N nutrition and plant defence is considered in terms of physical, biochemical and genetic mechanisms. Generally, N has negative effects on physical defences and the production of anti-microbial phytoalexins but positive effects on defence-related enzymes and proteins to affect local defence as well as systemic resistance. N nutrition can also influence defence via amino acid metabolism and hormone production to affect downstream defence-related gene expression via transcriptional regulation and nitric oxide (NO) production, which represents a direct link with N. Although the critical role of N nutrition in plant defences is stressed in this review, further work is urgently needed to provide a comprehensive understanding of how opposing virulence and defence mechanisms are influenced by interacting networks.

OsTSD2-mediated cell wall modification affects ion homeostasis and salt tolerance.

Salt stress is a major environmental threat to meeting the food demands of an increasing global population. The identification and exploitation of salt adaption mechanisms in plants are therefore vital for crop breeding. We here define the rice mutant (sstm1) whose salt sensitivity was unambiguously assigned to a single T-DNA insertion through segregational analysis following backcrossing to the wild type line. Insertion was within OsTSD2, which encoded a pectin methyltransferase. The sstm1 and allelic mutants, collectively known as tsd2, displayed higher content of Na+ and lower level of K+ in the shoot, which is likely to lead to reduced salt tolerance. Molecular analysis revealed reduced expression of the genes maintaining K+ /Na+ homeostasis in tsd2, including OsHKT1;5, OsSOS1, and OsKAT1. Furthermore, OsTSD2 influenced ion distribution between the hull and the rice seed, which could improve food safety with heavy metal pollution. Amino acid levels tended to be increased in tsd2 mutants, implicating a role of pectin in the regulation of metabolism. Taken together, we have demonstrated an important facet of salt tolerance, which implicated OsTSD2-mediated cell wall pectin modification as a key component that could be widely applied in crop science.

Exploring the Roles of Aquaporins in Plant⁻Microbe Interactions.

Aquaporins (AQPs) are membrane channel proteins regulating the flux of water and other various small solutes across membranes. Significant progress has been made in understanding the roles of AQPs in plants' physiological processes, and now their activities in various plant⁻microbe interactions are receiving more attention. This review summarizes the various roles of different AQPs during interactions with microbes which have positive and negative consequences on the host plants. In positive plant⁻microbe interactions involving rhizobia, arbuscular mycorrhizae (AM), and plant growth-promoting rhizobacteria (PGPR), AQPs play important roles in nitrogen fixation, nutrient transport, improving water status, and increasing abiotic stress tolerance. For negative interactions resulting in pathogenesis, AQPs help plants resist infections by preventing pathogen ingress by influencing stomata opening and influencing defensive signaling pathways, especially through regulating systemic acquired resistance. Interactions with bacterial or viral pathogens can be directly perturbed through direct interaction of AQPs with harpins or replicase. However, whilst these observations indicate the importance of AQPs, further work is needed to develop a fuller mechanistic understanding of their functions.