Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

Non-target impacts of soybean rust fungicides on the fungal entomopathogens of soybean aphid

Soybean aphid, Aphis glycines, has caused serious economic damage to soybean across the North Central US since its introduction to North America in 2000. The management of another invasive soybean pest, Asian soybean rust, Phakopsora pachyrhizi, using foliar fungicide applications has the potential to impact soybean aphid populations by suppressing beneficial fungal entomopathogens. In 2005 and 2006, we applied recommended soybean rust fungicide treatments, consisting of strobilurin and triazole fungicides, to small soybean plots in two locations to assess if such applications might suppress aphid fungal epizootics. In Lamberton, MN, in 2005, during the epizootic, fungicide-treated plots averaged 2.0 ± 0.7% (mean ± SE) disease prevalence while untreated plots averaged 14.2 ± 5.6%. In 2007, we applied strobilurin and strobilurin-triazole mix fungicides to single-plant microplots either before or after release of Pandora neoaphidis, the most commonly observed aphid pathogen in 2005 and 2006. Treatments that contained a mixture of two active ingredients significantly lowered peak and cumulative aphid disease prevalence in both early and late reproductive stage soybeans indicating that fungicide mixtures used to manage soybean rust can negatively impact an aphid-specific fungal pathogen. However, no consistent soybean aphid population response was observed in these studies of low levels of aphid fungal infection.     

Silicon‐Induced Changes in the Antioxidant System Reduce Soybean Resistance to Frogeye Leaf Spot

Frogeye leaf spot (FLS), caused by the fungus Cercospora sojina, is one of the most important soybean diseases and can cause great yield losses. Several studies have demonstrated that silicon (Si) enhances the plant antioxidant system, especially when they are subjected to stresses. Thus, this study was designed to evaluate the effect of Si on soybean resistance to FLS, on the antioxidant system, on the concentration of reactive oxygen species and on cellular damage during the infection process of C. sojina. Plants from cultivars Bossier and Conquista, susceptible and resistant to FLS, respectively, were supplied with either 0 (?Si) or 2 mm (+Si) and non‐inoculated or inoculated with C. sojina. FLS severity was greater for Bossier than for Conquista, regardless of the Si supply, and it was increased by Si for both cultivars. The activities of the most antioxidant enzymes were lower in the +Si plants than in the ?Si plants when they were not inoculated. Inoculated plants usually showed an increased enzyme activities and higher concentrations of ascorbate and reduced glutathione than did the non‐inoculated plants, regardless of Si supply. At advanced stages of fungal infection, the +Si‐inoculated plants from Bossier had higher activity of most antioxidant enzymes and higher concentrations of superoxide and malondialdehyde compared to the non‐inoculated plants as a result of an increased oxidative stress. The results from this study provide the first evidence that Si reduces the basal activity of antioxidant enzymes in soybean leaves leading to an increase in host susceptibility to FLS.     

Defence‐Related Enzymes in Soybean Resistance to Target Spot

Target spot, caused by the fungus Corynespora cassiicola, has become a serious foliar disease in soybean production in the Brazilian Cerrado. Information in the literature regarding the biochemical defence responses of soybean to C. cassiicola infection is rare. Therefore, the objective of this study was to determine the biochemical features associated with soybean resistance to target spot. The activities of chitinases (CHI), β‐1‐3‐glucanases (GLU), phenylalanine ammonia‐lyases (PAL), peroxidases (POX), polyphenol oxidases (PPO) and lipoxygenases (LOX), as well as the concentrations of total soluble phenolics (TSP) and lignin‐thioglycolic acid (LTGA) derivatives, were determined in soybean leaves from both a resistant (FUNDACEP 59) and a susceptible (TMG 132) cultivar. The target spot severity, number of lesions per cm² of leaflet and area under the disease progress curve were significantly lower for plants from cv. FUNDACEP 59 compared to plants from cv. TMG 132. The GLU, CHI, PAL, POX and PPO activities and the concentration of LTGA derivatives increased significantly, whereas LOX activity decreased significantly on the leaves infected by C. cassiicola. Inoculated plants from cv. FUNDACEP 59 showed a higher PPO activity and concentrations of TSP and LTGA derivatives at 4 and 6 days after inoculation compared to plants from cv. TMG 132. In conclusion, the results of this study demonstrated that the defence‐related enzyme activities increased upon C. cassiicola infection, regardless of the basal level of resistance of the cultivar studied. The increases in PPO activity and concentrations of TSP and LTGA derivatives, but lower LOX activity, at early stages of C. cassiicola infection were highly associated with soybean resistance to target spot.     

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE‐receptor kinase‐WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant‐parasitic cyst nematodes secrete CLE‐like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR‐LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode‐induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock‐down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.     

Leaf gas exchange and chlorophyll a fluorescence in soybean leaves infected by Phakopsora pachyrhizi

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most important foliar diseases affecting soybean production worldwide. This study aimed to investigate the photosynthetic performance (leaf gas exchange, chlorophyll (Chl) a fluorescence images and photosynthetic pigment pools) of soybean plants sprayed with Acibenzolar‐S‐Methyl (ASM) and the fungicide epoxiconazole + pyraclostrobin (Epo+Pyr) and further inoculated with P. pachyrhizi. The ASR symptoms progressed much faster on the leaves of plants from the control treatment (water spray) in comparison with the ASM and Epo+Pyr treatments. In general, the values for the leaf gas exchange parameters net carbon assimilation rate (A), stomatal conductance to water vapour (g_s), internal CO₂ concentration (C_i) and transpiration rate (E) increased for the infected plants sprayed with ASM or Epo+Pyr in comparison with plants from the control treatment. The values for the initial fluorescence (F_o), maximal fluorescence (F_m), maximal photosystem II quantum efficiency (F_v/F_m), effective photosystem II quantum yield (Y(II)) and quantum yield of regulated energy dissipation (Y(NPQ)) were consistently higher for the ASM and Epo+Pyr treatments in comparison with the control treatment at advanced stages of fungal infection. By contrast, the values for quantum yield of non‐regulated energy dissipation (Y(NO) were significantly lower for the ASM and Epo+Pyr treatments. The concentrations of total Chl a+b and carotenoids significantly increased for infected plants sprayed with ASM and Epo+Pyr in comparison with plants from the control treatment. The results of this study demonstrated that the spray of soybean plants with either ASM or Epo+Pyr contributed to reduce the negative effect of ASR on the photosynthesis of soybean plants.     

Soybean Resistance to Phakopsora pachyrhizi as Affected by Acibenzolar‐S‐Methyl,Jasmonic Acid and Silicon

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most important diseases on soybean. At the moment, ASR is managed mainly with fungicides due to the absence of commercial cultivars with resistance to this disease. This study evaluated the effects of acibenzolar‐Smethyl (ASM), jasmonic acid (JA), potassium silicate (PS) and calcium silicate (CS) on soybean resistance to ASR. The ASM, JA and PS were sprayed to leaves 24 h prior to inoculation with P. pachyrhizi. The CS was amended to the soil. The incubation period (time from the inoculation until symptoms development) was longer for plants growing in soil amended with CS or sprayed with ASM in comparison with plants sprayed with water (control). Plants sprayed with ASM had longer latent period (time from the inoculation until signs appearance) in comparison with the control plants. Plants sprayed with PS showed fewer uredia per cm² of leaf in relation to the control plants. The ASM and PS were the most effective treatments in reducing the ASR symptoms in contrast to the JA and CS treatments. The JA served as an inducer of susceptibility to ASR.     

The rice/maize pathogen Cochliobolus spp. infect and reproduce on Arabidopsis revealing differences in defensive phytohormone function between monocots and dicots

The fungal genus Cochliobolus describes necrotrophic pathogens that give rise to significant losses on rice, wheat, and maize. Revealing plant mechanisms of non‐host resistance (NHR) against Cochliobolus will help to uncover strategies that can be exploited in engineered cereals. Therefore, we developed a heterogeneous pathosystem and studied the ability of Cochliobolus to infect dicotyledons. We report here that C. miyabeanus and C. heterostrophus infect Arabidopsis accessions and produce functional conidia, thereby demonstrating the ability to accept Brassica spp. as host plants. Some ecotypes exhibited a high susceptibility, whereas others hindered the necrotrophic disease progression of the Cochliobolus strains. Natural variation in NHR among the tested Arabidopsis accessions can advance the identification of genetic loci that prime the plant’s defence repertoire. We found that applied phytotoxin‐containing conidial fluid extracts of C. miyabeanus caused necrotic lesions on rice leaves but provoked only minor irritations on Arabidopsis. This result implies that C. miyabeanus phytotoxins are insufficiently adapted to promote dicot colonization, which corresponds to a retarded infection progression. Previous studies on rice demonstrated that ethylene (ET) promotes C. miyabeanus infection, whereas salicylic acid (SA) and jasmonic acid (JA) exert a minor function. However, in Arabidopsis, we revealed that the genetic disruption of the ET and JA signalling pathways compromises basal resistance against Cochliobolus, whereas SA biosynthesis mutants showed a reduced susceptibility. Our results refer to the synergistic action of ET/JA and indicate distinct defence systems between Arabidopsis and rice to confine Cochliobolus propagation. Moreover, this heterogeneous pathosystem may help to reveal mechanisms of NHR and associated defensive genes against Cochliobolus infection.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense‐related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid‐targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild‐type (WT) counterparts. ROS build‐up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis‐related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast‐generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.     

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence‐related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full‐length cDNAs of GmSAMT1 from a SCN‐resistant soybean line and from a SCN‐susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli‐expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μm . To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN‐susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.     

Sub‐lethal UV‐C radiation induces callose,hydrogen peroxide and defence‐related gene expression in Arabidopsis thaliana

Exposure of plants to UV‐C irradiation induces gene expression and cellular responses that are commonly associated with wounding and pathogen defence, and in some cases can lead to increased resistance against pathogen infection. We examined, at a physiological, molecular and biochemical level, the effects of and responses to, sub‐lethal UV‐C exposure on Arabidopsis plants when irradiated with increasing dosages of UV‐C radiation. Following UV‐C exposure plants had reduced leaf areas over time, with the severity of reduction increasing with dosage. Severe morphological changes that included leaf glazing, bronzing and curling were found to occur in plants treated with the 1000 J·m^?2 dosage. Extensive damage to the mesophyll was observed, and cell death occurred in both a dosage‐ and time‐dependent manner. Analysis of H₂O₂ activity and the pathogen defence marker genes PR1 and PDF1.2 demonstrated induction of these defence‐related responses at each UV‐C dosage tested. Interestingly, in response to UV‐C irradiation the production of callose (β‐1,3‐glucan) was identified at all dosages examined. Together, these results show plant responses to UV‐C irradiation at much lower doses than have previously been reported, and that there is potential for the use of UV‐C as an inducer of plant defence.     

A Strobilurin Fungicide Relieves Bipolaris oryzae‐Induced Oxidative Stress in Rice

Although strobilurins are one of the most effective and broad spectrum classes of systemic fungicides, they may also increase plant stress tolerance by modulating the activity of antioxidant enzymes. To address this issue, the effect of azoxystrobin (Az) on the activity of antioxidant enzymes and on the concentrations of antioxidant metabolites and oxidative stress‐related compounds was studied in rice plants (cv. Metica‐1) either inoculated or not with Bipolaris oryzae, the causal agent of brown spot (BS). The Az minimally affected the enzyme activities, but consistently increased the glutathione reduced (GSH) concentrations in the noninoculated plants. The activities of superoxide dismutase, peroxidase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase and glutathione‐S‐transferase were increased upon B. oryzae infection, but such increases were greatly limited in the Az‐sprayed plants. Catalase activity decreased in the inoculated plants compared to the noninoculated plants regardless of fungicide treatment. The GSH concentration increased in response to the B. oryzae infection, and the Az‐sprayed plants sustained higher levels of GSH at advanced stages of fungal infection than did the nonsprayed plants. The inoculated plants exhibited an extensive oxidative stress as evidenced by higher concentrations of hydrogen peroxide and malondialdehyde compared to the noninoculated plants, but lower and later increases were recorded in the Az‐sprayed plants than in the nonsprayed plants. Therefore, Az greatly reduces B. oryzae‐induced oxidative stress by limiting BS development rather than by activating antioxidant enzymes. The GSH, however, seems to be Az‐modulated, and this may partially explain the constrained oxidative stress observed in the Az‐sprayed plants.