Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein

The expression patterns of plant defense genes encoding osmotin and osmotin-like proteins imply a dual function in osmotic stress and plant pathogen defense. We have produced transgenic potato (Solanum commersonii Dun.) plants constitutively expressing sense or antisense RNAs from chimeric gene constructs consisting of the cauliflower mosaic virus 35S promoter and a cDNA (pA13) for an osmotin-like protein. Transgenic potato plants expressing high levels of the pA13 osmotin-like protein showed an increased tolerance to the late-blight fungus Phytophthora infestans at various phases of infection, with a greater resistance at an early phase of fungal infection. There was a decrease in the accumulation of osmotin-like mRNAs and proteins when antisense transformants were challenged by fungal infection, although the antisense transformants did not exhibit any alterations in disease susceptibility. Expression of pA13 sense and antisense RNAs had no effect on the development of freezing tolerance in transgenic plants when assayed under a variety of conditions including treatments with abscisic acid or low temperature. These results provide evidence of antifungal activity for a potato osmotin-like protein against the fungus P. infestans, but do not indicate that pA13 osmotin-like protein is a major determinant of freezing tolerance.     

致病疫霉效应蛋白Pi16275的功能研究

[目的]分析致病疫霉效应蛋白Pi16275的超量表达对病原菌致病性的影响,明确Pi16275的亚细胞定位,筛选Pi16275在植物中的互作靶标蛋白及靶标蛋白在抵御病原菌侵染过程中的作用,初步揭示Pi16275在病原菌侵染植物过程中的作用机制.[方法]利用农杆菌介导的烟草瞬时表达系统在烟草叶片表皮细胞中瞬时表达Pi162...     

Transgenic potato plants overexpressing the pathogenesis-related STH-2 gene show unaltered susceptibility to Phytophthora infestans and potato virus X

The STH-2 gene is rapidly activated in potato leaves and tubers following elicitation or infection by Phytophthora infestans. However, its biochemical function remains unknown. In order to ascertain if STH-2 protein is directly involved in the defense of potato against pathogens, the STH-2 coding sequence under the control of the CaMV 35S promoter was introduced into potato plants. Transgenic plants expressing the STH-2 gene were analyzed for an altered pattern of susceptibility to a compatible race of P. infestans and to potato virus X. Results indicate that constitutive expression of the STH-2 gene did not reduce susceptibility of potato to these pathogens.     

Expression of the Phytophthora infestans ipiB and ipi0 genes in planta and in vitro

The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta. Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P. infestans infection. During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv. Moneymaker, the P. infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection. During the interaction with leaves of the partially resistant potato cv. Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed. Also in P. infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection. Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions. In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed. Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced. For ipiO gene expression, carbon deprivation appeared to be sufficient. The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression.     

Expression of the Phytophthora infestans ipiB and ipi0 genes in planta and in vitro

The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta. Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P. infestans infection. During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv. Moneymaker, the P. infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection. During the interaction with leaves of the partially resistant potato cv. Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed. Also in P. infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection. Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions. In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed. Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced. For ipiO gene expression, carbon deprivation appeared to be sufficient. The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression.     

An elicitor-and pathogen-induced cdna from potato encodes a stress-responsive cyclophilin

Differential mRNA display was used to identify pathogen-responsive, stress-related genes in potato cell suspensions treated with salicylic acid and a cell wall-derived elicitor from Phytophthora infestans. Among the positive clones identified, one was found to be expressed at a significantly higher level in elicited cells than in control cells. DNA sequencing of this amplicon revealed high homology and identified it as a potato cyclophilin cDNA. The maximum amount of the cyclophilin mRNA was found 9 to 12 h after elicitation. Cyclophilin (CyP) mRNA synthesis was also up-regulated from 12 to 24 h in potato leaves locally infected with zoospores from Phytophthora infestans. However, untreated leaves responding systemically to the pathogen showed only a weak, delayed response at 24 h post infection. The observed accumulation of potato CyP mRNA in response to salicylic acid, P. infestans elicitor and P. infestans infection, suggest that CyPs play an important role in plant stress responses.     

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.     

Functional Characterization of Dihydroflavonol-4-Reductase in Anthocyanin Biosynthesis of Purple Sweet Potato Underlies the Direct Evidence of Anthocyanins Function against Abiotic Stresses

Dihydroflavonol-4-reductase (DFR) is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam.) cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi) using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions.     

MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides

The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface.