Relationship between quantum efficiency of PSII and cold-induced plant resistance to fungal pathogens

The aim of the presented work was to study whether the efficiency of photosynthesis may influence resistance of hardened plants to disease. Seedlings of spring barley, meadow fescue and winter oilseed rape were chilled at 5 °C for 2, 4 or 6 weeks and at these deadlines the changes in cell membrane permeability (expressed as electrolyte leakage), chlorophyll fluorescence (initial fluorescence - F₀, maximal fluorescence - F_m, quantum yield of PSII - F_v/F_m) and net photosynthesis rate (F_N) were measured. Also, the influence of cold on the degree of plant resistance to economically important pathogens -Bipolaris sorokiniana or Phoma lingam was estimated. Two, four or six week-hardened plants were artificially infected: barley and fescue by B. sorokiniana, and oilseed rape by P. lingam. Hardening at 5 °C stimulated resistance of barley, fecue and rape to their specific pathogens. Six-week long acclimation was the most effective for plant resistance. Cold significantly changed cell membrane permeability and decreased chlorophyll fluorescence (F₀, F_m and F_v/F_m) of all studied plant species, while net photosynthesis rate was found to decrease only in barley. The results indicate that cold-induced resistance of plants to pathogens was correlated with a decrease in cell membrane permeability. In the case of fescue and barley a significant connection between the quantum yield of PSII and their resistance to B. sorokiniana was shown. Additionally, the resistance of barley to fungus was depended on net photosynthesis rate. In general this research shows that the efficiency of photosynthesis may be used as an indicator of plant resistance to disease.     

A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (M agnaporthe o ryzae re sistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.     

Transient over-expression of barley BAX Inhibitor-1 weakens oxidative defence and MLA12-mediated resistance to Blumeria graminis f.sp. hordei

BAX Inhibitor-1 (BI-1) is a conserved cell death suppressor protein. In barley, BI-1 ( HvBI-1 ) expression is induced upon powdery mildew infection and when over-expressed in epidermal cells of barley, HvBI-1 induces susceptibility to the biotrophic fungal pathogen Blumeria graminis . We co-expressed mammalian pro-apoptotic BAX together with HvBI-1, and the mammalian BAX antagonist BCL-X_L in barley epidermal cells. BAX expression led to cessation of cytoplasmic streaming and collapse of the cytoplasm while co-expression of HvBI-1 and BCL-X_L partially or completely, respectively, rescued cells from BAX lethality. When B. graminis was attacking epidermal cells, a green fluorescent protein fusion of HvBI-1 accumulated at the site of attempted penetration and was also present around haustoria. Over-expression of HvBI-1 in epidermal cells weakened a cell-wall-associated local hydrogen peroxide burst in a resistant mlo -mutant genotype and supported haustoria accommodation in race-specifically resistant MLA12 -barley. HvBI-1 is a cell death regulator protein of barley with the potential to suppress host defence reactions.     

Volatile-mediated signalling in barley induces metabolic reprogramming and resistance against the biotrophic fungus Blumeria hordei

• Plants have evolved diverse secondary metabolites to counteract biotic stress. Volatile organic compounds (VOCs) are released upon herbivore attack or pathogen infection. Recent studies suggest that VOCs can act as signalling molecules in plant defence and induce resistance in distant organs and neighbouring plants. However, knowledge is lacking on the function of VOCs in biotrophic fungal infection on cereal plants.
• We analysed VOCs emitted by 13 ± 1-day-old barley plants (Hordeum vulgare L.) after mechanical wounding using passive absorbers and TD-GC/MS. We investigated the effect of pure VOC and complex VOC mixtures released from wounded plants on the barley–powdery mildew interaction by pre-exposure in a dynamic headspace connected to a powdery mildew susceptibility assay. Untargeted metabolomics and lipidomics were applied to investigate metabolic changes in sender and receiver barley plants.
• Green leaf volatiles (GLVs) dominated the volatile profile of wounded barley plants, with (Z)-3-hexenyl acetate (Z3HAC) as the most abundant compound. Barley volatiles emitted after mechanical wounding enhanced resistance in receiver plants towards fungal infection. We found volatile-mediated modifications of the plant–pathogen interaction in a concentration-dependent manner. Pre-exposure with physiologically relevant concentrations of Z3HAC resulted in induced resistance, suggesting that this GLV is a key player in barley anti-pathogen defence.
• The complex VOC mixture released from wounded barley and Z3HAC induced e.g. accumulation of chlorophyll, linolenic acid and linolenate-conjugated lipids, as well as defence-related secondary metabolites, such as hordatines in receiving plants. Barley VOCs hence induce a complex physiological response and disease resistance in receiver plants.

     

Transcriptome-based analysis of resistance mechanisms to Bipolaris sorokiniana,a common wheat root-rot disease

• In common root and crown rot (CRR), Bipolaris sorokiniana (Sace.) is one of the important in wheat, causing considerable yield losses globally. Sources of resistance can provide a feasible and effective method of control for plant disease management. However, knowledge on mechanisms of resistance is scarce.
• We screened 33 wheat genotypes against B. sorokiniana under greenhouse and field conditions. In addition, real-time quantitative PCR (qPCR) analysis using ten novel candidate gene markers, Cre3, EDS1, LTP5, PGIP, PR-1, PIEP1, TLP, UGT, Stb6 and PFT, was conducted on leaves and roots, along with changes in activity of antioxidant enzymes, peroxidase, catalase, β-1,3-glucanase, and phenolic content for their involvement in disease impact mechanisms.
• Lowest disease severity was in ‘Alvand’, followed by ‘Baharan’ and ‘Bam’ as resistant genotypes. Quantitative gene expression showed that, although the candidate defence genes were upregulated 1.24- to 3.5-fold in wheat roots and leaves inoculated with B. sorokiniana, they were highly regulated in resistant varieties ‘Alvand’, ‘Mehregan’ and ‘Bam’. Cre3, a resistance gene to cereal cyst nematode Heterodera filipjevi, was regulated in cultivars resistant to B. sorokiniana. Similar results were obtained for Stb6, a gene resistant to Septoria tritici blotch, EDS1 resistant to powdery mildew, Blumeria graminis, and the genes PR-1 and UGT resistant to leaf rust, Puccinia triticina. Antioxidant enzyme activity also showed the highest increases in resistant genotypes.
• In conclusion, the T. aestivum–B. sorokiniana interaction in resistant wheat cultivars uses defence-related genes and enzymes that protect wheat towards sustainable development. Further such studies will shed light on simultaneous resistance to other diseases in wheat cultivars.

     

A novel effector,CsSp1, from Bipolaris sorokiniana,is essential for colonization in wheat and is also involved in triggering host immunity

The hemibiotrophic pathogen Bipolaris sorokiniana causes root rot, leaf blotching, and black embryos in wheat and barley worldwide, resulting in significant yield and quality reductions. However, the mechanism underlying the host–pathogen interactions between B. sorokiniana and wheat or barley remains unknown. The B. sorokiniana genome encodes a large number of uncharacterized putative effector proteins. In this study, we identified a putative secreted protein, CsSp1, with a classic N-terminal signal peptide, that is induced during early infection. A split-marker approach was used to knock out CsSP1 in the Lankao 9-3 strain. Compared with the wild type, the deletion mutant ∆Cssp1 displayed less radial growth on potato dextrose agar plates and produced fewer spores, and complementary transformation completely restored the phenotype of the deletion mutant to that of the wild type. The pathogenicity of the deletion mutant in wheat was attenuated even though appressoria still penetrated the host. Additionally, the infectious hyphae in the deletion mutant became swollen and exhibited reduced growth in plant cells. The signal peptide of CsSp1 was functionally verified through a yeast YTK12 secretion system. Transient expression of CsSp1 in Nicotiana benthamiana inhibited lesion formation caused by Phytophthora capsici. Moreover, CsSp1 localized in the nucleus and cytoplasm of plant cells. In B. sorokiniana-infected wheat leaves, the salicylic acid-regulated genes TaPAL, TaPR1, and TaPR2 were down-regulated in the ∆Cssp1 strain compared with the wild-type strain under the same conditions. Therefore, CsSp1 is a virulence effector and is involved in triggering host immunity.     

Influence of Seed‐borne Cochliobolus sativus (Anamorph Bipolaris sorokiniana) on Crown Rot and Root Rot of Barley and Wheat

The effect of seed‐borne pathogens of wheat and barley on crown and root rot diseases of seven barley cultivars (Jimah‐6, Jimah‐51, Jimah‐54, Jimah‐58, Omani, Beecher and Duraqi) and three wheat cultivars (Cooley, Maissani and Shawarir) was investigated. Bipolaris sorokiniana and Alternaria alternata were detected in seeds of at least eight cultivars, but Fusarium species in seeds of only two barley cultivars (Jimah‐54 and Jimah‐58). Crown rot and root rot symptoms developed on barley and wheat cultivars following germination of infected seeds in sterilized growing media. Bipolaris sorokiniana was the only pathogen consistently isolated from crowns and roots of the emerging seedlings. In addition, crown rot and root rot diseases of non‐inoculated barley cultivars correlated significantly with B. sorokiniana inoculum in seeds (P = 0.0019), but not with Fusarium or Alternaria (P > 0.05). These results indicate the role of seed‐borne inoculum of B. sorokiniana in development of crown rot and root rot diseases. Pathogenicity tests of B. sorokiniana isolates confirmed its role in inducing crown rot and root rot, with two wheat cultivars being more resistant to crown and root rots than most barley cultivars (P < 0.05). Barley cultivars also exhibited significant differences in resistance to crown rot (P < 0.05). In addition, black point disease symptoms were observed on seeds of three barley cultivars and were found to significantly affect seed germination and growth of some of these cultivars. This study confirms the role of seed‐borne inoculum of B. sorokiniana in crown and root rots of wheat and barley and is the first report in Oman of the association of B. sorokiniana with black point disease of barley.     

Functions of pipecolic acid on induced resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in tomato plants

Amino acid metabolic pathways are involved in the plant immune system. Pipecolic acid (Pip), a lysine-derived non-protein amino acid, acts as an important regulator of disease resistance. Here, we report the functions of Pip on tomato disease resistance. Tomato seedlings treated with 0.5 mM Pip showed increased resistance to Pst DC3000 and B. cinerea compared with the control. After pathogen infection, the expression of defence-related genes increased in plants pretreated with Pip, while reactive oxygen species (ROS) accumulation decreased. These data demonstrated that exogenous application of Pip induced resistance against Pst DC3000 and B. cinerea in tomatoes, possibly through the regulation of ROS accumulation and defence-related gene expression.     

Study of physio-biochemical responses elicited by potassium phosphite in downy mildew-infected cucumber plants

Potassium phosphite (KPhi) is widely used as a resistance inducer to protect plants against fungal pathogens. In the present study, the effect of KPhi on the activation of defence-related enzymes and biochemicals in Pseudoperonospora cubensis-challenged cucumber plants was investigated. Cucumber plants were treated with KPhi before or after inoculation with P. cubensis and leaf samples were collected at different time courses for physiological and biochemical assessments. Results revealed that the activity of reactive oxygen species (ROS)-scavenging enzymes like catalase, guaiacol peroxidase, superoxide dismutase and ascorbate peroxidase as well as proline and total carbohydrates contents were significantly increased by KPhi application, while hydrogen peroxide (H₂O₂) concentration, as a disease damage indicator was reduced. The maximum activity of ROS-scavenging system was achieved 3–4 days after KPhi application. These findings suggest that KPhi application prior to pathogen infection efficiently triggers plant defence responses which may reduce the disease severity.     

Time of day of leaf wounding determines plant biomass and affects the interplay between growth and defence in Brassica crops

• Recent discoveries pointed out the importance of the mutual correlation between timing of environmental stress and plant fitness. However, the internal reshaping of plant growth under daily stress sensing, and their metabolic coordination remain to be investigated. Thus, we studied the connection between time of day, growth and defence to understand how plant fitness is affected by diurnal stress inputs.
• We examined if simulated herbivory (leaf wounding) in the morning, at midday or the evening differentially influence plant defence state vs growth in three crop species of Brassica: broccoli (Brassica oleraceae), turnip greens (B. rapa) and rapeseed (B. napus).
• The data revealed that plant's tolerance of wounding stress is diurnally regulated in Brassica crops. Trade-offs between biomass and investment in glucosinolates (GSL) and phenolics were affected by timing of leaf stress. Negative correlations between biomass and induction of defence compounds were found for plants treeated in the morning and evening. However, the correlations were positive for midday treatment. Interestingly, we revealed a new connection between plant growth and changes in aliphatic GSLs and flavonoids in response to wounding.
• These data suggest that metabolic stress-dependent circadian oscillations in leaf defences could be one mechanism conferring a competitive advantage to plants to anticipate daily environmental variations by synchronizing them with growth. Moreover, this work provides first insights into how secondary metabolites are linked to growth response in a timing-related manner.

     

Green leaf volatiles and jasmonic acid enhance susceptibility to anthracnose diseases caused by Colletotrichum graminicola in maize

Colletotrichum graminicola is a hemibiotrophic fungus that causes anthracnose leaf blight (ALB) and anthracnose stalk rot (ASR) in maize. Despite substantial economic losses caused by these diseases, the defence mechanisms against this pathogen remain poorly understood. Several hormones are suggested to aid in defence against C. graminicola, such as jasmonic acid (JA) and salicylic acid (SA), but supporting genetic evidence was not reported. Green leaf volatiles (GLVs) are a group of well-characterized volatiles that induce JA biosynthesis in maize and are known to function in defence against necrotrophic pathogens. Information regarding the role of GLVs and JA in interactions with (hemi)biotrophic pathogens remains limited. To functionally elucidate GLVs and JA in defence against a hemibiotrophic pathogen, we tested GLV- and JA-deficient mutants, lox10 and opr7 opr8, respectively, for resistance to ASR and ALB and profiled jasmonates and SA in their stalks and leaves throughout infection. Both mutants were resistant and generally displayed elevated levels of SA and low amounts of jasmonates, especially at early stages of infection. Pretreatment with GLVs restored susceptibility of lox10 mutants, but not opr7 opr8 mutants, which coincided with complete rescue of JA levels. Exogenous methyl jasmonate restored susceptibility in both mutants when applied before inoculation, whereas methyl salicylate did not induce further resistance in either of the mutants, but did induce mutant-like resistance in the wild type. Collectively, this study reveals that GLVs and JA contribute to maize susceptibility to C. graminicola due to suppression of SA-related defences.     

Atmospheric CO2, plant nitrogen status and the susceptibility of plants to an acute increase in temperature

Abstract. Elevated levels of CO₂ in the atmosphere are expected to affect plant performance and may alter global temperature patterns. Changes in mean air temperatures that might be induced by rising levels of CO₂ and other greenhouse gases could also be accompanied by increased variability in daily temperatures such that acute increases in air temperature may be more likely than at present. Consequently, we investigated whether plants grown in a CO₂ enriched atmosphere would be differently affected by a heat shock than plants grown at ambient CO₂ levels. Plants of a C₃ annual (Abutilon theophrasti), a C₃ annual crop (Sinapis alba) and a C₄ annual (Amaranthus retroflexus) were grown from seed in growth chambers under either 400 or 700cm³ m^?3 CO₂, and were fertilized with either a high or low nutrient regime. Young seedlings of S. alba, as well as plants of all species in either the vegetative or reproductive phase of growth were exposed to a 4-h heat shock in which the temperature was raised an additional 14–23°C (depending on plant age). Total biomass and reproductive biomass were examined to determine the effect of CO₂, nutrient and heat shock treatments on plant performance. Heat shock, CO₂, and nutrient treatments, all had some significant effects on plant performance, but plants from both CO₂ treatments responded similarly to heat shocks. We also found, as expected, that plants grown under high CO₂ had dramatically decreased tissue N concentrations relative to plants grown under ambient conditions. We predicted that high-CO₂-grown plants would be more susceptible to a heat shock than ambient-CO₂-grown plants, because the reduced N concentrations of high-CO₂ grown plants could result in the reduced synthesis of heat shock proteins and reduced thermotolerance. Although we did not examine heat shock proteins, our results showed little relationship between plant nitrogen status and the ability of a plant to tolerate an acute increase in temperature.     

Phenylalanine Ammonia-lyase Activity in Barley After Infection with Bipolaris sorokiniana or Treatment with its Purified Xylanase

Phenylalanine ammonia-lyase (PAL) activity was determined from leaves and roots of two barley (Hordeum vulgare L.) cultivars after infection with a necrotrophic pathogen, Bipolaris sorokiniana (Sacc.) Shoem., and treatment with its purified xylanase. PAL activity increased in leaves of both cultivars 16 h after fungal inoculation but two phases, with activity peaks at 24–32 h and 40 h, were recorded only for the more resistant cultivar, Agneta. Attempts to use a PAL inhibitor, χ-amin, ooxyacetic acid, to increase susceptibility to B. sorokiniana in barley leaves were unsuccessful. Treatments of leaves with purified xylanase resulted in more rapid (4–12 h after injection), although reduced, induction of PAL compared with fungal injection. The higher the concentration of xylanase applied the earlier the activity peaks were detected. Fungal inoculation only slightly increased PAL activity in barley roots while xylanase treatment had no effect. The basal level of PAL was however much higher in roots than in leaves. In wheat, Triticum aestivum L. resistant to B. sorokiniana, the time-course of PAL induction after fungal infection and xylanase treatment resembled that for cv. Agneta, while in oats, Avena sativa L. (non-host) PAL activity did not change after the treatments. The results suggest that the second phase of PAL induction, associated only with responses of barley cv. Agneta and wheat, is linked with their resistance to B. sorokiniana infection. The possible role of xylanase as an elicitor of PAL is discussed.     

Plant defence suppression is mediated by a fungal sirtuin during rice infection by Magnaporthe oryzae

Crop destruction by the hemibiotrophic rice pathogen Magnaporthe oryzae requires plant defence suppression to facilitate extensive biotrophic growth in host cells before the onset of necrosis. How this is achieved at the genetic level is not well understood. Here, we report that a M. oryzae sirtuin, MoSir2, plays an essential role in rice defence suppression and colonization by controlling superoxide dismutase (SOD) gene expression. Loss of MoSir2 function in Δsir2 strains did not affect appressorial function, but biotrophic growth in rice cells was attenuated. Compared to wild type, Δsir2 strains failed to neutralize plant‐derived reactive oxygen species (ROS) and elicited robust defence responses in rice epidermal cells that included elevated pathogenesis‐related gene expression and granular depositions. Deletion of a SOD‐encoding gene under MoSir2 control generated Δsod1 deletion strains that mimicked Δsir2 for impaired rice defence suppression, confirming SOD activity as a downstream output of MoSir2. In addition, comparative protein acetylation studies and forward genetic analyses identified a JmjC domain‐containing protein as a likely target of MoSir2, and a Δsir2 Δjmjc double mutant was restored for MoSOD1 expression and defence suppression in rice epidermal cells. Together, this work reveals MoSir2 and MoJmjC as novel regulators of early rice cell infection.     

Specific patterns of changes in wheat gene expression after treatment with three antifungal compounds

The two fungicides azoxystrobin and fenpropimorph are used against powdery mildew and rust diseases in wheat (Triticum aestivumL). Azoxystrobin, a strobilurin, inhibits fungal mitochondrial respiration and fenpropimorph, a morpholin, represses biosynthesis of ergosterol, the major sterol of fungal membranes. Although the fungitoxic activity of these compounds is well understood, their effects on plant metabolism remain unclear. In contrast to the fungicides which directly affect pathogen metabolism, benzo(1,2,3) thiadiazole-7-carbothioic acid S-methylester (BTH) induces resistance against wheat pathogens by the activation of systemic acquired resistance in the host plant. In this study, we monitored gene expression in spring wheat after treatment with each of these agrochemicals in a greenhouse trial using a microarray containing 600 barley cDNA clones. Defence-related genes were strongly induced after treatment with BTH, confirming the activation of a similar set of genes as in dicot plants following salicylic acid treatment. A similar gene expression pattern was observed after treatment with fenpropimorph and some defence-related genes were induced by azoxystrobin, demonstrating that these fungicides also activate a defence reaction. However, less intense responses were triggered than with BTH. The same experiments performed under field conditions gave dramatically different results. No gene showed differential expression after treatment and defence genes were already expressed at a high level before application of the agrochemicals. These differences in the expression patterns between the two environments demonstrate the importance of plant growth conditions for testing the impact of agrochemicals on plant metabolism.     

Over-expression of the cell death regulator BAX inhibitor-1 in barley confers reduced or enhanced susceptibility to distinct fungal pathogens

BAX inhibitor-1 (BI-1) is a conserved cell death regulator protein that inhibits mammalian BAX-induced cell death in yeast, animals and plants. Additionally, HvBI-1 suppresses defense responses and resistance to the powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) when over-expressed in single epidermal cells of barley. To test the potential of ectopic expression of BI-1 to influence fungal interactions with crop plants, we produced stable transgenic barley plants expressing a green fluorescing protein (GFP) fusion of HvBI-1 under control of the cauliflower mosaic virus 35S promoter. GFP-HvBI-1 plants were fertile and did not display obvious developmental alterations when compared to wild type parents. GFP-HvBI-1 plants were more resistant to single cell death induced by ballistic delivery of a mammalian proapototic BAX expression construct and more susceptible to biotrophic Bgh. Microscopic observation of the interaction phenotype revealed that enhanced susceptibility, i.e. a higher degree of successful establishment of haustoria in epidermal cells, was associated with a reduced frequency of hypersensitive cell death reactions. In contrast, young seedlings of GFP-HvBI-1 barley were more resistant to Fusarium graminearum than wild type or azygous controls. Hence the effect of GFP-HvBI-1 on the outcome of a particular plant–fungus interaction appeared dependent on the lifestyle of the pathogen. V. Babaeizad and J. Imani contributed equally to this study.