Are the polygenic architectures of resistance to <Emphasis Type="Italic">Phytophthora capsici</Emphasis> and <Emphasis Type="Italic">P. parasitica</Emphasis> independent in pepper?

The pepper accession Criollo de Morelos 334 is the most efficient source of resistance currently known to Phytophthora capsici and P. parasitica. To investigate whether genetic controls of resistance to two Phytophthora species are independent, we compared the genetic architecture of resistance of CM334 to both Phytophthora species. The RIL population F5YC used to construct the high-resolution genetic linkage map of pepper was assessed for resistance to one isolate of each Phytophthora species. Inheritance of the P. capsici and P. parasitica resistance was polygenic. Twelve additive QTLs involved in the P. capsici resistance and 14 additive QTLs involved in the P. parasitica resistance were detected. The QTLs identified in this progeny were specific to these Phytophthora species. Comparative mapping analysis with literature data identified three colocations between resistance QTLs to P. parasitica and P. capsici in pepper. Whereas this result suggests presence of common resistance factors to the two Phytophthora species in pepper, which possibly derive from common ancestral genes, calculation of the colocation probability indicates that these colocations could occur by chance.     

Resistance to Phytophthora pathogens is dependent on gene silencing pathways in plants

RNA silencing is one of the main defence mechanisms employed by plants to fight pathogens. p19 protein encoded by the tomato bushy stunt virus (TBSVp19) is known as a suppressor of RNA silencing via siRNA sequestration to prevent the assembly of RISC. To better understand the impact of TBSVp19 on silencing and its roles in Phytophthora pathogens, we used the transient expression assay in Nicotiana benthamiana and found that the leaves expressing TBSVp19 were more susceptible to Phytophthora parasitica. Furthermore, we demonstrated that TBSVp19‐mediated plant susceptibility in N. benthamiana is dependent on RNA‐dependent RNA polymerase 6 (RDR6). We also tested the role of RNA silencing in resistance of soybean hairy roots to Phytophthora. The lesion size induced by P. sojae on TBSVp19‐expressing soybean hairy roots was slightly, but significantly larger than GFP‐expressing soybean hairy roots. Finally, the Arabidopsis gene silencing mutants ago1‐27, zip‐1, sgs3‐11 and rdr6‐11 were also examined for their resistance to P. parasitica. The results clearly showed that resistance levels of the mutants were visibly reduced compared with the wild type. Taken together, these results suggest that the gene silencing system in plants is essential for resistance to Phytophthora pathogens.     

Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway

Benzothiadiazole (BTH) is a novel chemical activator of disease resistance in tobacco, wheat and other important agricultural plants. In this report, it is shown that BTH works by activating SAR in Arabidopsis thaliana. BTH-treated plants were resistant to infection by turnip crinkle virus, Pseudomonas syringae pv ‘tomato’ DC3000 and Peronospora parasitica. Chemical treatment induced accumulation of mRNAs from the SAR-associated genes, PR-1, PR-2 and PR-5. BTH treatment induced both PR-1 mRNA accumulation and resistance against P. parasitica in the ethylene response mutants, etr1 and ein2, and in the methyl jasmonate-insensitive mutant, jar1, suggesting that BTH action is independent of these plant hormones. BTH treatment also induced both PR-1 mRNA accumulation and P. parasitica resistance in transgenic Arabidopsis plants expressing the nahG gene, suggesting that BTH action does not require salicylic acid accumulation. However, because BTH-treatment failed to induce either PR-1 mRNA accumulation or P. parasitica resistance in the non-inducible immunity mutant, nim1, it appears that BTH activates the SAR signal transduction pathway.     

Involvements of <Emphasis Type="Italic">S</Emphasis>-nitrosylation and denitrosylation in the production of polyphenols by <Emphasis Type="Italic">Inonotus obliquus</Emphasis>

Nitric oxide (NO) has been evidenced to mediate biosynthesis of polyphenols in Inonotus obliquus. However, it remains unknown how NO regulates their biosynthesis. Here we show that higher cellular NO levels coincided with higher accumulation of S-nitrosothiols (SNO; the products of NO combined with a specific residue in glutathione or proteins) and polyphenols, and higher activity of denitrosylated S-nitrosoglutathione reductase (GSNOR) and thioredoxin reductase (TrxR). This homeostasis was breached by GSNOR or TrxR inhibitors. Inhibiting GSNOR boosted TrxR activity, but reduced SNO formation, coinciding with an enhanced production of polyphenols. Likewise, inhibiting TrxR increased GSNOR activity and SNO production, but downregulated accumulation of polyphenols. Inhibiting GSNOR or TrxR also modified the polyphenolic profiles of I. obliquus. Suppressing GSNOR-enhanced biosynthesis of phelligridins C and H, inoscavin C and methyl inoscavin B, but reduced that of phelligridin D, methyl inoscavin A, davallialactone and methyl davallialactone, the typical polyphenols in I. obliquus. Similarly, downregulating TrxR increased production of phelligridin D, methyl inoscavin A, davallialactone, and methyl davallialactone, but shrinking that of phelligridins C and H, methyl inoscavin B and inoscavin C. Thus, in I. obliquus, the state of S-nitrosylation and denitrosylation affects not only the accumulation of polyphenols, but also their metabolic profiles.     

An RXLR effector secreted by Phytophthora parasitica is a virulence factor and triggers cell death in various plants

RXLR effectors encoded by Phytophthora species play a central role in pathogen–plant interactions. An understanding of the biological functions of RXLR effectors is conducive to the illumination of the pathogenic mechanisms and the development of disease control strategies. However, the virulence function of Phytophthora parasitica RXLR effectors is poorly understood. Here, we describe the identification of a P. parasitica RXLR effector gene, PPTG00121 (PpE4), which is highly transcribed during the early stages of infection. Live cell imaging of P. parasitica transformants expressing a full-length PpE4 (E4FL)-mCherry protein indicated that PpE4 is secreted and accumulates around haustoria during plant infection. Silencing of PpE4 in P. parasitica resulted in significantly reduced virulence on Nicotiana benthamiana. Transient expression of PpE4 in N. benthamiana in turn restored the pathogenicity of the PpE4-silenced lines. Furthermore, the expression of PpE4 in both N. benthamiana and Arabidopsis thaliana consistently enhanced plant susceptibility to P. parasitica. These results indicate that PpE4 contributes to pathogen infection. Finally, heterologous expression experiments showed that PpE4 triggers non-specific cell death in a variety of plants, including tobacco, tomato, potato and A. thaliana. Virus-induced gene silencing assays revealed that PpE4-induced cell death is dependent on HSP90, NPK and SGT1, suggesting that PpE4 is recognized by the plant immune system. In conclusion, PpE4 is an important virulence RXLR effector of P. parasitica and recognized by a wide range of host plants.     

Potato StMPK7 is a downstream component of StMKK1 and promotes resistance to the oomycete pathogen Phytophthora infestans

A cascade formed by phosphorylation events of mitogen-activated protein kinases (MAPKs) takes part in plant stress responses. However, the roles of these MAPKs in resistance of potato (Solanum tuberosum) against Phytophthora pathogens is not well studied. Our previous work showed that a Phytophthora infestans RXLR effector targets and stabilizes the negative regulator of MAPK kinase 1 of potato (StMKK1). Because in Arabidopsis thaliana the AtMPK4 is the downstream phosphorylation target of AtMKK1, we performed a phylogenetic analysis and found that potato StMPK4/6/7 are closely related and are orthologs of AtMPK4/5/11/12. Overexpression of StMPK4/7 enhances plant resistance to P. infestans and P. parasitica. Yeast two-hybrid analysis revealed that StMPK7 interacts with StMKK1, and StMPK7 is phosphorylated on flg22 treatment and by expressing constitutively active StMKK1 (CA-StMKK1), indicating that StMPK7 is a direct downstream signalling partner of StMKK1. Overexpression of StMPK7 in potato enhances potato resistance to P. infestans. Constitutively active StMPK7 (CA-StMPK7; StMPK7^{D198G, E202A}) was found to promote immunity to Phytophthora pathogens and to trigger host cell death when overexpressed in Nicotiana benthamiana leaves. Cell death triggered by CA-StMPK7 is SGT1/RAR1-dependent. Furthermore, cell death triggered by CA-StMPK7 is suppressed on coexpression with the salicylate hydroxylase NahG, and StMPK7 activation promotes salicylic acid (SA)-responsive gene expression. We conclude that potato StMPK7 is a downstream signalling component of the phosphorelay cascade involving StMKK1 and StMPK7 plays a role in immunity to Phytophthora pathogens via an SA-dependent signalling pathway.     

Strategies of attack and defense in plant–oomycete interactions, accentuated for Phytophthora parasitica Dastur (syn. P. Nicotianae Breda de Haan)

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to their particular physiological characteristics, no efficient treatments against diseases caused by these microorganisms are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. Available data are scarce, and genomic approaches were mainly developed for the two species, Phytophthora infestans and Phytophthora sojae. However, these two species are exceptions from, rather than representative species for, the genus. P. infestans is a foliar pathogen, and P. sojae infects a narrow range of host plants, while the majority of Phytophthora species are quite unselective, root-infecting pathogens. To represent this majority, Phytophthora parasitica emerges as a model for the genus, and genomic resources for analyzing its interaction with plants are developing. The aim of this review is to assemble current knowledge on cytological and molecular processes that are underlying plant–pathogen interactions involving Phytophthora species and in particular P. parasitica, and to place them into the context of a hypothetical scheme of co-evolution between the pathogen and the host.     

Immunological Discrimination of Phytophthora cinnamomi from other Phytophthorae Pathogenic on Chestnut

A polyclonal antiserum (A379) against water soluble proteins from Phytophthora cinnamomi mycelium was produced in rabbit. In ELISA, the 1 : 10 000 diluted antiserum revealed only Phytophthora isolates, not allowing a clear‐cut discrimination among congenerous species, in spite of a generally higher reactivity on P. cinnamomi proteins. The antiserum gave positive reactions in Western blot analyses against mycelial proteins from nine species of Phytophthora and Pythium sp. (grown on rich media), but not with Rhizoctonia solani, binucleate Rhizoctonia, Verticillium dahliae, Fusarium oxysporum and Cryphonectria parasitica. All Phytophthora species showed common epitopes on proteins of molecular masses 77, 66, 51 and 48 kDa. However, a species‐specific protein of 55 kDa was immunodecorated only in P. cinnamomi samples, thus allowing univocal identification of this species. When tested against total proteins from the same fungi grown on water, the antibody revealed diagnostic bands of 55 and 51 kDa in P. cinnamomi only. The antiserum is therefore suitable for the specific identification of P. cinnamomi emerging in distilled water from infected tissues of chestnut, blueberry and azalea.     

The Raf-like kinase Raf36 negatively regulates plant resistance against the oomycete pathogen Phytophthora parasitica by targeting MKK2

Oomycetes represent a unique group of plant pathogens that are phylogenetically distant from true fungi and cause significant crop losses and environmental damage. Understanding of the genetic basis of host plant susceptibility facilitates the development of novel disease resistance strategies. In this study, we report the identification of an Arabidopsis thaliana T-DNA mutant with enhanced resistance to Phytophthora parasitica with an insertion in the Raf-like mitogen-activated protein kinase kinase kinase gene Raf36. We generated additional raf36 mutants by CRISPR/Cas9 technology as well as Raf36 complementation and overexpression transformants, with consistent results of infection assays showing that Raf36 mediates Arabidopsis susceptibility to P. parasitica. Using a virus-induced gene silencing assay, we silenced Raf36 homologous genes in Nicotiana benthamiana and demonstrated by infection assays the conserved immune function of Raf36. Mutagenesis analyses indicated that the kinase activity of Raf36 is important for its immune function and interaction with MKK2, a MAPK kinase. By generating and analysing mkk2 mutants and MKK2 complementation and overexpression transformants, we found that MKK2 is a positive immune regulator in the response to P. parasitica infection. Furthermore, infection assay on mkk2 raf36 double mutant plants indicated that MKK2 is required for the raf36-conferred resistance to P. parasitica. Taken together, we identified a Raf-like kinase Raf36 as a novel plant susceptibility factor that functions upstream of MKK2 and directly targets it to negatively regulate plant resistance to P. parasitica.     

Starch utilization by Phytophthora spp

Summary Phytophthora spp. were grown on artificial starch agar medium. In some cases, the capacity of starch utilization could be a useful tool in species separation ofPhytophthora. Based on the ability to hydrolyse starch,P. palmivora andP. parasitica could be readily distinguished, whereasP. parasitica andP. parasitica var.nicotianae, P. megasperma andP. megasperma var.sojae (P. sojae) behaved similarly. Starch hydrolysis was indicated by a clear unstained zone within the fungal colony when treated with iodine solution. Simple quantitative analysis of starch hydrolysis was made feasible by the following formula:Starch Hydrolysis Index (S.H.I.) = Mean diameter of clear starch hydrolysis zone (d) / Mean diameter of fungal colony (D)     

Phytophthora Species Infecting Hardy Ornamentals in Nurseries and the Managed Environment in Scotland

Between 2002 and the end of 2009, more than 4000 samples from hardy ornamental plants, collected in surveys for Phytophthora ramorum, were examined to establish the occurrence and diversity of Phytophthora species in Scotland. The samples were gathered from more than 77 plant genera in nurseries, gardens and amenity landscapes. Fifteen different Phytophthora spp. were isolated and identified either by polymerase chain reaction (PCR) or by sequencing of the ITS1, 5.8S subunit and ITS2 region of the ribosomal RNA gene. The most widespread Phytophthora spp. were P. ramorum and P. syringae, followed by P. cactorum, P. kernoviae, P. plurivora, P. cambivora, P. citrophthora, P. taxon ‘Pgchlamydo’, P. pseudosyringae and some single isolates of P. cinnamomi, P. cryptogea, P. gonapodyides, P. nicotianae and P. hibernalis. One isolate did not match any known species. In relation to the number of samples, Phytophthora was found more frequently in trade premises than in gardens or amenity landscapes and the species diversity was higher, highlighting the risks involved in plant trade.     

Characterization and pathogenicity of three Phytophthora spp. recovered from rivers in Bulgaria

Many Phytophthora species are pathogens on fruit trees and may cause destructive diseases. In the current study, we examined six Phytophthora isolates recovered from rivers in Bulgaria, representatives of the following three species: Phytophthora chlamydospora, P. pseudocryptogea and P. syringae. Morphological traits, cardinal temperatures and growth rates of the isolates were described. We found considerable variation in the size of sporangia and significant difference in the mycelial growth rates of the two P. pseudocryptogea isolates, along with multiple polymorphic sites in the ITS region of one of them. In the cases of the other two Phytophthora species, no such differences were found between the isolates. Both P. chlamydospora isolates had a lower optimum growth temperature compared with the reported in the literature for this species. In pathogenicity tests against leaves and fruits of apple, pear, cherry, apricot and plum, the isolates proved to be capable of causing infections with varying severity. P. chlamydospora showed to be the most aggressive towards the leaves, while P. pseudocryptogea isolates induced the highest percentage of decay on the fruits of all tested tree species, which may suggest partial organ or tissue specificity. The demonstrated infection capacity of the described isolates points out the investigated Phytophthora species as a potential threat for the orchards in Bulgaria, if favourable conditions are available.     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

Distribution of Phytophthora citrophthora R.E. Sm. and E.H. Sm. and P. parasitica Dastur within citrus orchards in Kerman province,Iran

In this research, distribution of Phytophthora species were determined in Kerman Province (Bam, Shahdad and Arzuiyeh) during 2004–2007. The Phytophthora species were isolated from infected root, crown and soil. Root and crown pieces were washed and cultured on a CMA-PARPH medium. The isolation from infected soil was performed by bating method using citrus leaves. It was identified by morphological and several physiological characteristics. Distribution of species was determined by recording the number of isolates recovered from samples from each city. In this study, from 220 soil samples collected from 52 citrus orchards, 80 isolates of Phytophthora were recovered. Among of all isolates of Phytophthora, P. parasitica and P. citrophthora were the most important species of causal agent of gummosis on Citrus sp. Distribution of P. citrophthora was highest in Arzuiyeh and lowest in Bam and Shahdad cities, while distribution of P. parasitica was highest in Bam and Shahdad cities. The majority of soil samples contained only P. parasitica, but a few of percentage samples containing P. citrophthora alone and both of fungi in cites samples.     

Ectopic expression of <Emphasis Type="Italic">Dahlia merckii</Emphasis> defensin <Emphasis Type="Italic">DmAMP</Emphasis>1 improves papaya resistance to <Emphasis Type="Italic">Phytophthora palmivora</Emphasis> by reducing pathogen vigor

Phytophthora spp., some of the more important casual agents of plant diseases, are responsible for heavy economic losses worldwide. Plant defensins have been introduced as transgenes into a range of species to increase host resistance to pathogens to which they were originally susceptible. However, the effectiveness and mechanism of interaction of the defensins with Phytophthora spp. have not been clearly characterized in planta. In this study, we expressed the Dahlia merckii defensin, DmAMP1, in papaya (Carica papaya L.), a plant highly susceptible to a root, stem, and fruit rot disease caused by Phytophthora palmivora. Extracts of total leaf proteins from transformed plants inhibited growth of Phytophthora in vitro and discs cut from the leaves of transformed plants inhibited growth of Phytophthora in a bioassay. Results from our greenhouse inoculation experiments demonstrate that expressing the DmAMP1 gene in papaya plants increased resistance against P. palmivora and that this increased resistance was associated with reduced hyphae growth of P. palmivora at the infection sites. The inhibitory effects of DmAMP1 expression in papaya suggest this approach has good potential to impart transgenic resistance against Phytophthora in papaya.     

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.     

Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD⁺ as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD⁺, the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD⁺ and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.