NtTPN1: a RPP8‐like R gene required for Potato virus Y‐induced veinal necrosis in tobacco

Potato virus Y (PVY) is one of the most damaging viruses of tobacco. In particular, aggressive necrotic strains (PVY^N) lead to considerable losses in yield. The main source of resistance against PVY is linked to the va locus. However, va‐overcoming PVY isolates inducing necrotic symptoms were observed in several countries. In this context, it is important to find va‐independent protection strategies. In a previous study, the phenotyping of 162 tobacco varieties revealed 10 accessions that do not carry the va allele and do not exhibit typical PVY^N‐induced veinal necrosis. Despite the absence of necrotic symptoms, normal viral accumulation in these plants suggests a va‐independent mechanism of tolerance to PVY^N‐induced systemic veinal necrosis. Fine mapping of the genetic determinant(s) was performed in a segregating F2 population. The tolerance trait is inherited as a single recessive gene, and allelism tests demonstrated that eight of the 10 tolerant varieties carry the same determinant. Anchoring the linkage map to the tobacco genome physical map allowed the identification of a RPP8‐like R gene, called NtTPN1 (for t abacum P VY‐induced 相似文献   

RNA-dependent RNA polymerase 1 delays the accumulation of viroids in infected plants

RNA-dependent RNA polymerase 1 (RDR1) is essential for plant antiviral defence, but its role in plant defence against viroid infection remains unknown. The present study aimed to identify the function and mechanism of RDR1 in plant resistance to viroid infection. Overexpression of Nicotiana tabacum RDR1 (NtRDR1) delayed the accumulation of potato spindle tuber viroid (PSTVd) genomic RNA and PSTVd-derived small RNA (sRNA) in Nicotiana benthamiana plants at the early invasion stage, but not in the late stage of infection. Conversely, virus-induced gene silencing of tomato RDR1 (SlRDR1a) increased the susceptibility to PSTVd infection (increased viroid accumulation). Salicylic acid (SA) pretreatment induced SlRDR1a expression and enhanced the defence against PSTVd infection in tomato plants. Our study demonstrated that RDR1 is involved in SA-mediated defence and restricts the early systemic invasion by PSTVd in plants. The decreased PSTVd accumulation in N. benthamiana was not caused by efficient accumulation of PSTVd sRNAs. These results deepen our understanding of the mechanism of RDR1 in plant defence responses to viroid attack.     

Involvement of Hydrogen Peroxide, Peroxidase and Superoxide Dismutase in Response of Medicago truncatula Lines Differing in Susceptibility to Phoma medicaginis Infection

This study was undertaken to assess the involvement of hydrogen peroxide (H₂O₂), peroxidase (POX; EC 1.11.1.7) and superoxide dismutase (SOD; EC 1.15.1.1) in Medicago truncatula in relation with susceptibility to Phoma medicaginis infection. Several M. truncatula lines were studied in terms of their response to P. medicaginis infection. Fifteen days after inoculation (dai), differences in susceptibility were found. DZA45.5 was the least susceptible line and F83005.5 was the most susceptible line. Microscopic analysis of fungal development was performed in inoculated detached leaves of the DZA45.5 and F83005.5 lines. No significant difference was observed in events from conidia germination to penetration. Differences became apparent during the colonization process as the pathogen was able to sporulate rapidly increasing its concentration on the tissue of F83005.5 in comparison with DZA45.5. To characterize the susceptibility of the two lines, histochemical detection of H₂O₂ was made in detached leaves. H₂O₂ detection showed an early accumulation of this component in cells of DZA45.5 at 1 dai. However, H₂O₂ was detected in few, if any, cells in the tissues of the most susceptible line, F83005.5. The activity of POX and SOD were determined spectrophotometrically in leaves of intact inoculated plants of both lines. Phoma medicaginis inoculation of DZA45.5 and F83005.5 did not affect POX activity level in leaves when compared with control uninoculated plants. SOD activity showed a significant decrease in F83005.5 and DZA45.5 leaves at 4 dai and 9 dai, respectively, in comparison with control plants. In control plants POX activity was significantly higher in the least susceptible line DZA45.5 in comparison with F83005.5. Early and higher production of H₂O₂ and elevated basal POX activity in cells of the least susceptible line, DZA45.5 could explain its ability to be less favourable to the colonization and reproduction of P. medicaginis in comparison with the most susceptible line, F83005.5.     

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.     

Strong resistance to potato tuber necrotic ringspot disease in potato induced by transformation with coat protein gene sequences from an NTN isolate of Potato virus Y

The potato cv. Igor is susceptible to infection with Potato virus Y (PVY) and in Slovenia it has been so severely affected with NTN isolates of PVY causing potato tuber necrotic ringspot disease (PTNRD) that its cultivation has ceased. Plants of cv. Igor were transformed with two transgenes that contained coat protein gene sequence of PVY^NTN. Both transgenes used PVY sequence in a sense (+) orientation, one in native translational context (N‐CP), and one with a frame‐shift mutation (FS‐CP). Although most transgenic lines were susceptible to infection with PVY^NTN and PVY^O, several lines showed resistance that could be classified into two types. Following manual or graft inoculation, plants of partially resistant lines developed some symptoms in foliage and tubers, and virus titre in the foliage, estimated by ELISA, was low or undetectable. In highly resistant (R) lines, symptoms did not develop in foliage and on tubers, and virus could not be detected in foliage by ELISA or infectivity assay. Four lines from 34 tested (two N‐CP and two FS‐CP) were R to PVY^NTN and PVY^O and one additional line was R to PVY^O. When cv. Spey was transformed with the same constructs, they did not confer strong resistance to PVY^O.     

Comparison of transmission efficiency of various isolates of Potato virus Y among three aphid vectors

Potato virus Y (PVY) strains are transmitted by different aphid species in a non‐persistent, non‐circulative manner. Green peach aphid (GPA), Myzus persicae Sulzer, is the most efficient vector in laboratory studies, but potato aphid (PA), Macrosiphum euphorbiae Thomas (both Hemiptera: Aphididae, Macrosiphini), and bird cherry‐oat aphid (BCOA), Rhopalosiphum padi L. (Hemiptera: Aphididae, Aphidini), also contribute to PVY transmission. Studies were conducted with GPA, PA, and BCOA to assess PVY transmission efficiency for various isolates of the same strain. Treatments included three PVY strains (PVY^O, PVY^N:O, PVY^NTN) and two isolates of each strain (Oz and NY090031 for PVY^O; Alt and NY090004 for PVY^N:O; N4 and NY090029 for PVY^NTN), using each of three aphid species as well as a sham inoculation. Virus‐free tissue‐cultured plantlets of potato cv. Russet Burbank were used as virus source and recipient plants. Five weeks post inoculation, recipient plants were tested with quantitative DAS‐ELISA to assess infection percentage and virus titer. ELISA‐positive recipient plants were assayed with RT‐PCR to confirm presence of the expected strains. Transmission efficiency (percentage infection of plants) was highest for GPA, intermediate for BCOA, and lowest for PA. For all aphid species, transmission efficiency did not differ significantly between isolates within each strain. No correlations were found among source plant titer, infection percentage, and recipient plant titer. For both GPA and BCOA, isolates of PVY^NTN were transmitted with greatest efficiency followed by isolates of PVY^O and PVY^N:O, which might help explain the increasing prevalence of necrotic strains in potato‐growing regions. Bird cherry‐oat aphid transmitted PVY with higher efficiency than previously reported, suggesting that this species is more important to PVY epidemiology than has been considered.     

Jasmonoyl‐l‐isoleucine hydrolase 1 (JIH1) regulates jasmonoyl‐l‐isoleucine levels and attenuates plant defenses against herbivores

For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA‐Ile permanently inactivates JA‐Ile‐mediated signaling in plants, the alternative deactivation pathway of JA‐Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl‐l ‐isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA‐Ile and IAA‐Ala in vitro. When the herbivory‐inducible NaJIH1 gene was silenced by RNA interference, JA‐Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild‐type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild‐type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA‐Ile‐dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory‐elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA‐Ile‐hydroxylation and carboxylation of JA‐Ile, rapidly attenuates the JA‐Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature.     

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

Expression of Coat Protein of an Ordinary Strain of Potato virus Y in Escherichia coli and Production of Polyclonal Antibodies for Diagnosis

The coat protein gene (CP) of an ordinary strain of Potato virus Y (PVY^O) was cloned into the expression vector, pET‐28a(+). The insert was sequenced and analysis showed that the CP gene was in frame with intact N‐terminal 6X histidine tags. An approximately 35 kDa recombinant fusion protein was observed in inclusion bodies of induced Escherichia coli BL21 cells. This fusion protein was purified and used as antigen to raise polyclonal antibodies in rabbits. In Western blot and dot blot immuno‐binding assay (DIBA), both PVY^O‐CP IgG and PVY^O IgG strongly reacted with the recombinant CP. The PVY^O‐CP IgG could detect PVY^O in infected samples up to 1 : 3200 dilutions. A PVY^O‐CP ELISA kit was prepared and compared with conventional ELISA kit based on purified virus particles (PVY^O ELISA kit). The PVY^O‐CP ELISA kit consistently detected the PVY^O in DAS‐ELISA of field samples and was as effective as PVY^O ELISA kit.     

ZxAKT1 is essential for K+ uptake and K+/Na+ homeostasis in the succulent xerophyte Zygophyllum xanthoxylum

The inward‐rectifying K⁺ channel AKT1 constitutes an important pathway for K⁺ acquisition in plant roots. In glycophytes, excessive accumulation of Na⁺ is accompanied by K⁺ deficiency under salt stress. However, in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits excellent adaptability to adverse environments, K⁺ concentration remains at a relatively constant level despite increased levels of Na⁺ under salinity and drought conditions. In this study, the contribution of ZxAKT1 to maintaining K⁺ and Na⁺ homeostasis in Z. xanthoxylum was investigated. Expression of ZxAKT1 rescued the K⁺‐uptake‐defective phenotype of yeast strain CY162, suppressed the salt‐sensitive phenotype of yeast strain G19, and complemented the low‐K⁺‐sensitive phenotype of Arabidopsis akt1 mutant, indicating that ZxAKT1 functions as an inward‐rectifying K⁺ channel. ZxAKT1 was predominantly expressed in roots, and was induced under high concentrations of either KCl or NaCl. By using RNA interference technique, we found that ZxAKT1‐silenced plants exhibited stunted growth compared to wild‐type Z. xanthoxylum. Further experiments showed that ZxAKT1‐silenced plants exhibited a significant decline in net uptake of K⁺ and Na⁺, resulting in decreased concentrations of K⁺ and Na⁺, as compared to wild‐type Z. xanthoxylum grown under 50 mm NaCl. Compared with wild‐type, the expression levels of genes encoding several transporters/channels related to K⁺/Na⁺ homeostasis, including ZxSKOR, ZxNHX, ZxSOS1 and ZxHKT1;1, were reduced in various tissues of a ZxAKT1‐silenced line. These findings suggest that ZxAKT1 not only plays a crucial role in K⁺ uptake but also functions in modulating Na⁺ uptake and transport systems in Z. xanthoxylum, thereby affecting its normal growth.