Detoxification of sesquiterpene phytoalexins byGibberella pulicaris (Fusarium sambucinum) and its importance for virulence on potato tubers

Summary Gibberella pulicaris (Fusarium sambucinum) is a major cause of dry-rot of stored potatoes (Solanum tuberosum) worldwide. The ability of field strains ofG. pulicaris to cause dry-rot is correlated with their ability to detoxify sesquiterpene phytoalexins produced by potato. All highly virulent field strains can detoxify the sesquiterpenes rishitin and lubimin. Meiotic recombinational analysis indicates that rishitin detoxification can be controlled at two or more loci. High virulence has been associated with one of these loci, designatedRiml. Detoxification of rishitin and lubimin comprises a complex pattern of reactions involving epoxidation, dehydrogenation, and cyclization. To date, seven lubimin metabolites and one rishitin metabolite have been characterized. Genes for rishitin and lubimin detoxification are being cloned fromG. pulicaris in order to more rigorously analyze the role and regulation of sesquiterpene metabolism in potato dry-rot. Our results indirectly support a role for sesquiterpene phytoalexins in resistance of potato tubers to dry-rot and may enhance research on alternative control strategies for this economically important potato disease.     

An ABC transporter and a cytochrome P450 of Nectria haematococca MPVI are virulence factors on pea and are the major tolerance mechanisms to the phytoalexin pisatin

The fungal plant pathogen Nectria haematococca MPVI produces a cytochrome P450 that is responsible for detoxifying the phytoalexin pisatin, produced as a defense mechanism by its host, garden pea. In this study, we demonstrate that this fungus also produces a specific ATP-binding cassette (ABC) transporter, NhABC1, that enhances its tolerance to pisatin. In addition, although both mechanisms individually contribute to the tolerance of pisatin and act as host-specific virulence factors, mutations in both genes render the fungus even more sensitive to pisatin and essentially nonpathogenic on pea. NhABC1 is rapidly induced after treatment with pisatin in vitro and during infection of pea plants. Furthermore, NhABC1 was able to confer tolerance to the phytoalexin rishitin, produced by potato. NhABC1 appears to be orthologous to GpABC1 of the potato pathogen Gibberella pulicaris and, along with MoABC1 from Magnaporthe oryzae, resides in a phylogenetically related clade enriched with ABC transorters involved in virulence. We propose that NhABC1 and the cytochrome P450 may function in a sequential manner in which the energy expense from pisatin efflux by NhABC1 releases the repression of the cytochrome P450, ultimately allowing pisatin tolerance by two mechanisms. These results demonstrate that a successful pathogen has evolved multiple mechanisms to overcome these plant antimicrobial compounds.     

The effect of rishitin on potato tonoplast vesicle and vacuole proton transport

Rishitin, a known potato phytoalexin, was tested for its effects on proton transport. Like the pterocarpan phytoalexins, glyceollin and phaseollin, rishitin was found to inhibit proton transport. At 100 μM rishitin, proton transport in potato tonoplast vesicles was inhibited by >95%. This inhibition appears to be due to an increase in proton conductance and not to inhibition of the tonoplast ATPase. Potato vacuoles were also shown to have increased proton leakage in the presence of rishitin.     

Effect of mycorrhization on the accumulation of rishitin and solavetivone in potato plantlets challenged with<Emphasis Type="Italic"> Rhizoctonia solani</Emphasis>

The effect of colonization with the vesicular-arbuscular mycorrhizal fungus Glomus etunicatum on the content of rishitin and solavetivone was determined in potato plants cv. Goldrush challenged with Rhizoctonia solani. Mycorrhization stimulated significantly the accumulation of both phytoalexins in roots of plantlets challenged with R. solani but did not influence phytoalexin levels in non-challenged plantlet roots. No accumulation of solavetivone or rishitin was detected in shoots. In Petri dish bioassays, rishitin and solavetivone inhibited mycelial growth of R. solani.     

Genetic Analysis of the Role of Phytoalexin Detoxification in Virulence of the Fungus Nectria haematococca on Chickpea (Cicer arietinum)

Chickpea (Cicer arietium L.) produces the antimicrobial compounds (phytoalexins) medicarpin and maackiain in response to infection by microorganisms. Nectria haematococca mating population (MP) VI, a fungus pathogenic on chickpea, can metabolize maackiain and medicarpin to less toxic products. These reactions are thought to be detoxification mechanisms in N. haematococca MP VI and required for pathogenesis by this fungus on chickpea. In the present study, these hypotheses were tested by examining the phenotypes of progeny from crosses of the fungus that segregated for genes (Mak genes) controlling phytoalexin metabolism. Mak1 and Mak2, two genes that individually confer the ability to convert maackiain to its 1a-hydroxydienone derivative, were linked to higher tolerance of the phytoalexins and high virulence on chickpea. These results indicate that this metabolic reaction is a mechanism for increased phytoalexin tolerance in the fungus, which thereby allows a higher virulence on chickpea. Mak3, a gene conferring the ability to convert maackiain to its 6a-hydroxypterocarpan derivative, also increased tolerance to maackiain in strains which carried it; however, the contribution of Mak3 to the overall level of pathogenesis could not be evaluated because most progeny from the cross segregating for this gene were low in virulence. Thus, metabolic detoxification of phytoalexins appeared to be necessary, as demonstrated in the Mak1 and Mak2 crosses, but not sufficient by itself, as in the Mak3 cross, for high virulence of N. haematococca MP VI on chickpea.     

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.     

Comparisons between Phytoalexin Concentrations and the Extent of Rotting of Potato Tubers Inoculated with Erwinia carotovora sub sp. atroseptica, E. carotovora sub sp. carotovora or E. chrysanthemi

Isolates of Erwinia carotovora sub sp. atroseptica, E. carotovora sub sp. carotovora and E. chrysanthemi were compared for their ability to rot potato tubers and to elicit the accumulation of phytoalexins. With three incubation temperatures (15, 22 and 30°C) tubers were more susceptible at higher temperatures and mean rot weights were inversely correlated with mean rishitin concentrations. However, within treatment variation between tubers in the extent of rotting was not correlated with rishitin concentration suggesting that other factor(s) also affect resistance. Phytoalexins, including 15-dihydrolubimin, 3-hydroxylubimin, phytuberin and solavetivone, were measured by high pressure liquid chromatography using a silica column.     

MgAtr7, a new type of ABC transporter from Mycosphaerella graminicola involved in iron homeostasis

The ABC transporter-encoding gene MgAtr7 from the wheat pathogen Mycosphaerella graminicola was cloned based upon its high homology to ABC transporters involved in azole-fungicide sensitivity. Genomic and cDNA sequences indicated that the N-terminus of this ABC transporter contains a motif characteristic for a dityrosine/pyoverdine biosynthesis protein. This makes MgAtr7 the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. A homologue of MgAtr7 containing the same biosynthetic moiety was only found in the Fusarium graminearum genome and not in any other fungal genome examined so far. The gene structure of both orthologous transporters is highly conserved and the genomic area surrounding the ABC transporter exhibits micro-synteny between M. graminicola and F. graminearum. Functional analyses revealed that MgAtr7 is neither required for virulence nor involved in fungicide sensitivity but indicated a role in maintenance of iron homeostasis.     

An in vitro control mechanism for potato stress metabolite biosynthesis

Ethylene/oxygen (E/O₂) elevates sesquiterpenoid stress metabolite (SSM) levels in potato (Solanum tuberosum L.) tuber tissue which is reacting hypersensitively. To determine whether E/O₂ retards SSM turnover, a measured amount of rishitin was applied to tuber tissue which was then incubated in air or E/O₂, and rishitin disappearance was monitored. No difference in the rate of rishitin disappearance was detected between air and E/O₂ incubations. However, tissue treated with rishitin and incubated in E/O₂ accumulated intermediates of the katahdinone and phytuberin pathways. This was not the case in rishitin-air treatments. These results suggest the dual involvement of ethylene and SSM intermediates in the regulation of the biosynthesis of SSM, compounds which may serve as phytoalexins.     

The ybiT gene of Erwinia chrysanthemi codes for a putative ABC transporter and is involved in competitiveness against endophytic bacteria during infection

We investigated the role in bacterial infection of a putative ABC transporter, designated ybiT, of Erwinia chrysanthemi AC4150. The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, as well as structural similarity with proteins of Streptomyces spp. involved in resistance to macrolide antibiotics. The gene contiguous to ybiT, designated as pab (putative antibiotic biosynthesis) showed sequence similarity with Pseudomonas and Streptomyces genes involved in the biosynthesis of antibiotics. A ybiT mutant (BT117) was constructed by marker exchange. It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria. These results indicate that the ybiT gene plays a role in the in planta fitness of the bacteria.     

The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.     

猪链球菌2型ABC转运蛋白gene0910敲除突变体的构建及活性分析

目的:构建猪链球菌2型(Streptococcus suis type 2)强毒株05ZYH3389K毒力岛上的ABC转运蛋白gene0910敲除突变体,并初步分析其活性,为进一步研究猪链球菌假想毒力因子在致病中的作用提供实验基础。方法:以猪链球菌2型05ZYH33基因组为模板,扩增gene0910两侧各约500bp左右的片段为上下游同源臂,以pSET1质粒为模板,扩增氯霉素抗性基因Cm为中间片段,采用重叠PCR方法搭建三个片段,并克隆到自杀载体pSET4S上,构建基因敲除的载体。电转化05ZYH33感受态细胞,经30℃双交换和40℃质粒丢失,最后点板法筛选出基因敲除突变体△0910。对突变株和野生株的生物学活性及小鼠的致病性进行了初步比较。结果:PCR分析和测序结果均显示gene0910完全被氯霉素抗性基因Cm所替代,基因敲除突变体构建成功。结论:突变株的生物学活性和对小鼠的致病性与野生株相比差异不显著。     

The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea , a necrotrophic pathogen of A. thaliana . Exposure of B. cinerea to camalexin induces expression of BcatrB , an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3 , cyp71A13 , pad3 or pad2 , and was strongly reduced in ups1 . Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.     

GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments.     

Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium

In response to iron deprivation, Salmonella enterica serovar Typhimurium secretes two catecholate-type siderophores, enterobactin and its glucosylated derivative salmochelin. Although the systems responsible for enterobactin synthesis and acquisition are well characterized, the mechanisms of salmochelin secretion and acquisition, as well as its role in Salmonella virulence, are incompletely understood. Herein we show by liquid chromatography-mass spectrometry analysis of culture supernatants from wild type and isogenic mutant bacterial strains that the Major Facilitator Superfamily pump EntS is the major exporter of enterobactin and the ABC transporter IroC exports both salmochelin and enterobactin. Growth promotion experiments demonstrate that IroC is not required for utilization of Fe-enterobactin or Fe-salmochelin, as had been previously suggested, but the ABC transporter protein FepD is required for utilization of both siderophores. Salmonella mutants deficient in salmochelin synthesis or secretion exhibit reduced virulence during systemic infection of mice.     

The ABC transporters in Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram‐negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter‐related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In‐depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid‐like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Effects of Controlled Atmospheres on Production of Sesquiterpenoid Stress Metabolites by White Potato Tuber: Possible Involvement of Cyanide-resistant Respiration

Levels of katahdinone (solavetivone), lubimin, rishitin, and phytuberin, sesquiterpenoid stress metabolites of white potato (Solanum tuberosum), were monitored in tuber slices which were challenged with an extract of Phytophthora infestans and incubated under controlled atmospheres. A mixture of ethylene in air enhanced stress metabolite production. This enhancement was amplified by higher partial pressures of oxygen. Stress metabolite production was inhibited by salicylhydroxamic acid. These results suggest the involvement of cyanide-resistant respiration in the production of potato stress metabolites, compounds which may serve as phytoalexins.