A key enzyme for flavin synthesis is required for nitric oxide and reactive oxygen species production in disease resistance

Nitric oxide (NO) and reactive oxygen species (ROS) play key roles in plant immunity. However, the regulatory mechanisms of the production of these radicals are not fully understood. Hypersensitive response (HR) cell death requires the simultaneous and balanced production of NO and ROS. In this study we indentified NbRibA encoding a bifunctional enzyme, guanosine triphosphate cyclohydrolase II/3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase, which participates in the biosynthesis of flavin, by screening genes related to mitogen‐activated protein kinase‐mediated cell death, using virus‐induced gene silencing. Levels of endogenous riboflavin and its derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are important prosthetic groups for several enzymes participating in redox reactions, decreased in NbRibA‐silenced Nicotiana benthamiana. Silencing NbRibA compromised not only HR cell death, but also the NO and ROS production induced by INF1 elicitin and a constitutively active form of NbMEK2 (NbMEK2^DD), and also induced high susceptibility to oomycete Phytophthora infestans and ascomycete Colletotrichum orbiculare. Compromised radical production and HR cell death induced by INF1 in NbRibA‐silenced leaves were rescued by adding riboflavin, FMN or FAD. These results indicate that flavin biosynthesis participates in regulating NO and ROS production, and HR cell death.     

Different responses of two tobacco cultivars and their cell suspension cultures to quercinin,a novel elicitin from Phytophthora quercina

In this study we describe the response of two tobacco cultivars (Nicotiana tabacum L. cv. Bel B and Bel W3) and their cell suspension cultures to quercinin, a novel elicitin produced by the oak pathogen Phytophthora quercina. N-terminal sequencing of the purified protein proved that it belongs to the basic β-elicitins with threonine on position 13. Both tobacco leaves and cells of the cultivar Bel W3 showed hypersensitive cell death after quercinin treatment. Leaves of Bel B also developed quercinin-induced necrosis but higher concentrations of quercinin were necessary as compared to Bel W3. Also Bel B cells showed cell death induction only at the highest quercinin concentration (20 nM). In cell suspension experiments we also measured the quercinin-induced oxidative burst, which occurred in both cultivars. H₂O₂ production in Bel B increased with increasing quercinin concentration and was inhibited only at the highest elicitin concentration (20 nM) whereas the oxidative burst in Bel W3 was completely abolished by 5 nM quercinin. Furthermore we demonstrated that neither H₂O₂ nor superoxide were responsible for cell death induction since neither the inhibitor diphenyleneiodonium (DPI) nor the enzymes catalase (CAT) and superoxide dismutase (SOD) influenced the hypersensitive reaction (HR) in Bel W3 cells. Due to the different response of Bel W3 and Bel B towards the P. quercina elicitin, our system represents an interesting tool to elucidate signaling pathways in tobacco leading to hypersensitive cell death.     

Modification of disease resistance of tobacco callus tissues by cytokinins

The effects of differing cytokinin and auxin concentrations on resistance of tobacco (Nicotiana tabacum L.) tissue cultures to race 0 of Phytophthora parasitica var. nicotianae were examined. With 1 micromolar kinetin and either 11.5 micromolar indoleacetic acid or 1 micromolar 2,4-dichlorophen-oxyacetic acid, tissues from resistant cultivars exhibited a “hypersensitive” reaction to zoospores of the fungus and subsequently were colonized only slightly. With susceptible cultivars or with tissues from resistant cultivars supplied with higher cytokinin levels (e.g. 10 micromolar kinetin), this hypersensitive reaction did not occur and tissues were heavily colonized. Benzylaminopurine and kinetin were particularly effective in eliminating both the hypersensitive reaction and disease resistance. Zeatin and 6-(3-methyl-2-butenylamino)purine were less effective. Increases in indoleacetic acid levels reversed the effects of high cytokinin concentrations. The balance of phytohormones apparently controls the host response to the fungus; thus, in this system, resistance or susceptibility can be studied without changing either host or fungal genotype.     

Alternaria on cruciferous plants. 4.Alternaria species on seed of some cruciferous crops and their pathogenicity

The incidence ofAlternaria spp. on seed samples of cruciferous vegetable crops was surveyed between 1990 and 1992. Some commercial seed lots of crucifers which are commonly grown in Japan were infested withAlternaria species. ThreeAlternaria species were encountered on the seed samples ofBrassica campestris, B. orelacea, andRaphanus sativus. The most frequently detected species wereA. japonica andA. alternata onB. campestris, A. brassicicola onB. oleracea, andA. japonica andA. alternata onR. sativus, respectively.Alternaria brassicae was not detected in this study.Alternaria brassicicola isolates from these crops produced necrotic lesions on all of the crucifer seedlings inoculated, whileA. japonica induced different reactions in different plants or plant parts depending on isolates used in inoculation tests. In contrast, most isolates ofA. alternata could not produce necrotic lesions on foliage leaves of crucifers inoculated, although some of them produced clear lesions only on cotyledons.Alternaria alternata associated with these cruciferous crop seeds was considered to be an oppotunistic parasite of these crops.     

Suppression of Cdc27B expression induces plant defence responses

Non-host resistance is the most general form of disease resistance in plants because it is effective against most phytopathogens. The importance of hypersensitive responses (HRs) in non-host resistance of Nicotiana species to the oomycete Phytophthora is clear. INF1 elicitin, an elicitor obtained from the late-blight pathogen Phytophthora infestans , is sufficient to induce a typical HR in Nicotiana species. The molecular mechanisms that underlie the non-host resistance component of plant defence responses have been investigated using differential-display polymerase chain reaction (PCR) in a model HR system between INF1 elicitin and tobacco BY-2 cells. Differential-display PCR has revealed that Cdc27B is down-regulated in tobacco BY-2 cells after treatment with INF1 elicitin. Cdc27B is one of 13 essential components of the anaphase-promoting complex or cyclosome (APC/C)-type E3 ubiquitin ligase complex in yeast. This APC/C-type E3 ubiquitin ligase complex regulates G2-to-M phase transition of the cell cycle by proteolytic degradation. In this study, we investigated the roles of this gene, NbCdc27B , in plant defence responses using virus-induced gene silencing. Suppression of NbCdc27B in Nicotiana benthamiana plants induced defence responses and a gain of resistance to Colletotrichum lagenarium fungus. Elicitin-induced hypersensitive cell death (HCD) was inhibited mildly in plants silenced with tobacco rattle virus::Cdc27B. Cdc27B could manage the signalling pathways of plant defence responses as a negative regulator without HCD.     

The phytoalexin camalexin induces fundamental changes in the proteome of Alternaria brassicicola different from those caused by brassinin

Camalexin is the major phytoalexin produced by Alternaria thaliana, but is absent in Brassica species that usually produce phytoalexin blends containing brassinin and derivatives. The protein profiles of A. brassicicola treated with camalexin were evaluated using proteomics and metabolic analyses and compared with those treated with brassinin. Conidial germination and mycelial growth of A. brassicicola in liquid media amended with camalexin and brassinin showed that fungal growth was substantially slower in presence of camalexin than brassinin; chemical analyses revealed that A. brassicicola detoxified camalexin at much slower rate than brassinin. Two-dimensional gel electrophoresis (2-DE) followed by tryptic digestion and capillary liquid chromatography-mass spectrometric analyses identified 158 different proteins, of which 45 were up-regulated and 113 were down-regulated relative to controls. Venn diagram analyses of differentially expressed proteins in cultures of A. brassicicola incubated with camalexin and brassinin indicated clear differences in the effect of each phytoalexin, with camalexin causing down-regulation of a larger number of proteins than brassinin. Overall, results of this work suggest that each phytoalexin has several different targets in the cells of A. brassicicola, and that camalexin appears to have greater potential to protect cultivated Brassica species against A. brassicicola than brassinin.     

Distribution and Expression of Elicitin Genes in the Interspecific Hybrid Oomycete Phytophthora alni

Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3′ untranslated region (3′UTR)-specific PCRs and sequencing. The occurrence of multiple 3′UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.     

Potential Role of Elicitins in the Interaction between Phytophthora Species and Tobacco

The potential role of extracellular elicitor proteins (elicitins) from Phytophthora species as avirulence factors in the interaction between Phytophthora and tobacco was examined. A survey of 85 Phytophthora isolates representing 14 species indicated that production of elicitin is almost ubiquitous except for isolates of Phytophthora parasitica from tobacco. The production of elicitins by isolates of P. parasitica correlated without exception with low or no virulence on tobacco. Genetic analysis was conducted by using a cross between two isolates of P. parasitica, segregating for production of elicitin and virulence on tobacco. Virulence assays of the progeny on tobacco confirmed the correlation between production of elicitin and low virulence.     

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca²⁺ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca²⁺ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca²⁺ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.     

Biosynthesis and Secretion of Cryptogein, a Protein Elicitor Secreted by Phytophthora cryptogea

The phytopathogenic fungi Phytophthora subspecies elicit hypersensitive-like necroses on their nonhost tobacco (Nicotiana tabacum), with the exception of the tobacco pathogen Phytophthora nicotianæ. In culture, these fungi—except P. nicotianæ—secrete proteins, called elicitins, that cause these remote leaf necroses and are responsible for the incompatible reaction. These proteins protect tobacco against invasion by the agent of the tobacco black shank, P. nicotianæ, which is unable to produce such an elicitor. Cryptogein, secreted by Phytophthora cryptogea, has been purified, sequenced, and characterized as an elicitin, a novel family of 10 kilodalton holoproteins. In the present paper, we examined the secretion and biosynthesis of this protein elicitor from P. cryptogea culture. Results showed that the secretion of cryptogein began later than its synthesis and stopped earlier, simultaneously with mycelium growth, when the nitrogen source in the culture medium was nearly exhausted. Electrophoretic patterns of total protein from mycelium extracts and N-terminal sequence analysis showed that cryptogein accumulated in the mycelium in its mature form. The comparison of the immunoselected in vitro translation products with ³⁵S in vivo-labeled cryptogein showed that cryptogein was synthesized as a preprotein with a signal peptide removed cotranslationally before the secretion into the culture medium. Immunoselected in vitro-synthesized products were subjected to radiosequencing to clearly determine the N-terminal position and the size (20 amino acids) of the signal peptide. Cryptogein did not undergo any other posttranslational modification.     

Involvement of the caleosin/peroxygenase RD20 in the control of cell death during Arabidopsis responses to pathogens

Caleosins, mostly found in lipid droplets of seeds and leaves, are believed to play physiological roles through their enzymatic capacities to produce oxylipins. We recently identified the caleosin RD20 as a peroxygenase reducing endogenous fatty acid hydroperoxides into their corresponding alcohols. Such oxylipins confer tolerance to oxidative stress by decreasing reactive oxygen species accumulation and by minimizing cell death. RD20 expression being induced by pathogens, we have examined the mode of action of this caleosin in response to biotic stress. Plants overexpressing RD20 exhibited an alteration of their leaf cuticle wax components and an increased resistance to the fungus Alternaria brassicicola. Conversely, silencing RD20 led to an enhanced propagation of the fungus and to reduced severity of the damages caused by the inoculation of the bacteria Pseudomonas syringae pv tomato. We discuss these findings and propose that the major function of RD20 is to generate oxylipins modulating oxidative status and cell death.     

Genetic analysis of hybrid seed formation ability of Brassica rapa in intergeneric crossings with Raphanus sativus

A hybridization barrier leads to the inability of seed formation after intergeneric crossings between Brassica rapa and Raphanus sativus. Most B. rapa lines cannot set intergeneric hybrid seeds because of embryo breakdown, but a B. rapa line obtained from turnip cultivar ‘Shogoin-kabu’ is able to produce a large number of hybrid seeds as a maternal parent by crossings with R. sativus. In ‘Shogoin-kabu’ crossed with R. sativus, developments of embryos and endosperms were slower than those in intraspecific crossings, but some of them grew to mature seeds without embryo breakdown. Intergeneric hybrid seeds were obtained in a ‘Shogoin-kabu’ line at a rate of 0.13 per pollinated flower, while no hybrid seeds were obtained in a line developed from Chinese cabbage cultivar ‘Chiifu’. F₁ hybrid plants between the lines of ‘Shogoin-kabu’ and ‘Chiifu’ set a larger number of hybrid seeds per flower, 0.68, than both the parental lines. Quantitative trait loci (QTLs) for hybrid seed formation were analyzed after intergeneric crossings using two different F₂ populations derived from the F₁ hybrids, and three QTLs with significant logarithm of odds scores were detected. Among them, two QTLs, i.e., one in linkage group A10 and the other in linkage group A01, were detected in both the F₂ populations. These two QTLs had contrary effects on the number of hybrid seeds. Epistatic interaction between these two QTLs was revealed. Possible candidate genes controlling hybrid seed formation ability in QTL regions were inferred using the published B. rapa genome sequences.     

Phytophthora sojae apoplastic effector AEP1 mediates sugar uptake by mutarotation of extracellular aldose and is recognized as a MAMP

Diseases caused by Phytophthora pathogens devastate many crops worldwide. During infection, Phytophthora pathogens secrete effectors, which are central molecules for understanding the complex plant–Phytophthora interactions. In this study, we profiled the effector repertoire secreted by Phytophthora sojae into the soybean (Glycine max) apoplast during infection using liquid chromatography–mass spectrometry. A secreted aldose 1-epimerase (AEP1) was shown to induce cell death in Nicotiana benthamiana, as did the other two AEP1s from different Phytophthora species. AEP1 could also trigger immune responses in N. benthamiana, other Solanaceae plants, and Arabidopsis (Arabidopsis thaliana). A glucose dehydrogenase assay revealed AEP1 encodes an active AEP1. The enzyme activity of AEP1 is dispensable for AEP1-triggered cell death and immune responses, while AEP-triggered immune signaling in N. benthamiana requires the central immune regulator BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1. In addition, AEP1 acts as a virulence factor that mediates P. sojae extracellular sugar uptake by mutarotation of extracellular aldose from the α-anomer to the β-anomer. Taken together, these results revealed the function of a microbial apoplastic effector, highlighting the importance of extracellular sugar uptake for Phytophthora infection. To counteract, the key effector for sugar conversion can be recognized by the plant membrane receptor complex to activate plant immunity.

Phytophthora sojae apoplastic effector AEP1 triggers pattern-triggered immunity in nonhost plants and contributes to P. sojae virulence by promoting the uptake of extracellular sugar.     

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five β-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel β-barrel structure. However, the β-strands were found to display a unique topology, one pair of these β-strands formed a parallel β-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern–triggered immunity in N. benthamiana.     

Five Xanthomonas type III effectors suppress cell death induced by components of immunity-associated MAP kinase cascades

Mitogen-activated protein kinase (MAPK) cascades play a fundamental role in signaling of plant immunity and mediate elicitation of cell death. Xanthomonas spp. manipulate plant signaling by using a type III secretion system to deliver effector proteins into host cells. We examined the ability of 33 Xanthomonas effectors to inhibit cell death induced by overexpression of components of MAPK cascades in Nicotiana benthamiana plants. Five effectors inhibited cell death induced by overexpression of MAPKKKα and MEK2, but not of MAP3Kϵ. In addition, expression of AvrBs1 in yeast suppressed activation of the high osmolarity glycerol MAPK pathway, suggesting that the target of this effector is conserved in eukaryotic organisms. These results indicate that Xanthomonas employs several type III effectors to suppress immunity-associated cell death mediated by MAPK cascades.     

Hyperspectral and Thermal Imaging of Oilseed Rape (Brassica napus) Response to Fungal Species of the Genus Alternaria

In this paper, thermal (8-13 µm) and hyperspectral imaging in visible and near infrared (VNIR) and short wavelength infrared (SWIR) ranges were used to elaborate a method of early detection of biotic stresses caused by fungal species belonging to the genus Alternaria that were host (Alternaria alternata, Alternaria brassicae, and Alternaria brassicicola) and non-host (Alternaria dauci) pathogens to oilseed rape (Brassica napus L.). The measurements of disease severity for chosen dates after inoculation were compared to temperature distributions on infected leaves and to averaged reflectance characteristics. Statistical analysis revealed that leaf temperature distributions on particular days after inoculation and respective spectral characteristics, especially in the SWIR range (1000-2500 nm), significantly differed for the leaves inoculated with A. dauci from the other species of Alternaria as well as from leaves of non-treated plants. The significant differences in leaf temperature of the studied Alternaria species were observed in various stages of infection development. The classification experiments were performed on the hyperspectral data of the leaf surfaces to distinguish days after inoculation and Alternaria species. The second-derivative transformation of the spectral data together with back-propagation neural networks (BNNs) appeared to be the best combination for classification of days after inoculation (prediction accuracy 90.5%) and Alternaria species (prediction accuracy 80.5%).     

The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b

Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3b^P132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3b^P132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a “helper” that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.     

Molecular mechanisms of generation for nitric oxide and reactive oxygen species, and role of the radical burst in plant immunity

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and H₂O₂ accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.