Salmonella Typhimurium-specific bacteriophage ΦSH19 and the origins of species specificity in the Vi01-like phage family

Background

PHYVV and PepGMV are plant viruses reported in Mexico and Southern US as causal agents of an important pepper disease known as "rizado amarillo". Mixed infections with PHYVV and PepGMV have been reported in several hosts over a wide geographic area. Previous work suggested that these viruses might interact at the replication and/or movement level in a complex manner. The aim of present report was to study some aspects of a synergistic interaction between PHYVV and PepGMV in pepper plants. These include analyses of symptom severity, viral DNA concentration and tissue localization of both viruses in single and mixed infections.

Results

Mixed infections with PepGMV and PHYVV induced symptoms more severe than those observed in single viral infections. Whereas plants infected with either virus (single infection) presented a remission stage with a corresponding decrease in viral DNA levels, double-infected plants did not present symptom remission and both viral DNA concentrations dramatically increased. In situ hybridization experiments revealed that both viruses are restricted to the vascular tissue. Interestingly, the amount of viral DNA detected was higher in plants inoculated with PepGMV than that observed in PHYVV-infected plants. During mixed infections, the location of both viruses remained similar to the one observed in single infections, although the number of infected cells increases. Infections with the tripartite mixture PHYVV (A+B) + PepGMV A produced a similar synergistic infection to the one observed after inoculation with both full viruses. On the contrary, tripartite mixture PepGMV (A+B) + PHYVV A did not produce a synergistic interaction. In an attempt to study the contribution of individual genes to the synergism, several mutants of PHYVV or PepGMV were inoculated in combination with the corresponding wild type, second virus (wt PepGMV or wt PHYVV). All combinations tested resulted in synergistic infections, with exception of the TrAP mutant of PepGMV (PepGMV TrAP-) + PHYVV.

Conclusion

In this report, we have demonstrated that synergistic interaction between PHYVV and PepGMV during a mixed infection is mainly due to an increased DNA concentration of both viruses, without any noticeable effect on the localization of either virus on infected plant tissue. Our results have shown that the viral component A from PepGMV is important for synergism during PHYVV-PepGMV mixed infections.     

抗病基因Bdv2抑制大麦黄矮病毒复制和运动的分子证据

小麦-中间偃麦草易位系YW642含有一个源于中间偃麦草7X染色体的抗性基因Bdv2,对大麦黄矮病毒GAV株系具有高度抗性。为有效控制该病毒和阐明抗黄矮病机制,采用半定量RT-PCR的方法,研究了大麦黄矮病毒GAV株系在YW642及其感病姊妹系YW641中积累浓度的差异。分别在接种病毒不同时间、不同部位上取样,用半定量RT-PCR的方法来检测GAV的积累浓度。在接种部位,抗病植株中病毒的浓度远远低于感病植株。在侵染的前5d,抗病植株YW642中病毒会有一定程度的复制和积累,但随后病毒浓度开始下降,接种14—16d时没有检测到病毒;而在感病株系中,病毒积累的浓度远远高于抗病植株,并一直维持一个较高的浓度。在未接种部位．感病植株中可检测到较高浓度的病毒,说明病毒能从接种点很快运动到未接种部位,并大量复制。而在抗病系YW642中,未接种部位始终未检测到病毒。实验结果从分子水平上证明,在抗病植株中BYDV的复制和运动均受到了极大的抑制：这是抗病基因Bdv2与BYDV互作后,激活了一系列防御基因的结果。另外还确定了防御基因诱导表达的时间,为从抗病植株中分离抗病相关基因、研究抗黄矮病机制提供了取样的依据。     

Transcriptional Response of Virus-Infected Cassava and Identification of Putative Sources of Resistance for Cassava Brown Streak Disease

Cassava (Manihot esculenta) is a major food staple in sub-Saharan Africa, which is severely affected by cassava brown streak disease (CBSD). The aim of this study was to identify resistance for CBSD as well as to understand the mechanism of putative resistance for providing effective control for the disease. Three cassava varieties; Kaleso, Kiroba and Albert were inoculated with cassava brown streak viruses by grafting and also using the natural insect vector the whitefly, Bemisia tabaci. Kaleso expressed mild or no disease symptoms and supported low concentrations of viruses, which is a characteristic of resistant plants. In comparison, Kiroba expressed severe leaf but milder root symptoms, while Albert was susceptible with severe symptoms both on leaves and roots. Real-time PCR was used to estimate virus concentrations in cassava varieties. Virus quantities were higher in Kiroba and Albert compared to Kaleso. The Illumina RNA-sequencing was used to further understand the genetic basis of resistance. More than 700 genes were uniquely overexpressed in Kaleso in response to virus infection compared to Albert. Surprisingly, none of them were similar to known resistant gene orthologs. Some of the overexpressed genes, however, belonged to the hormone signalling pathways and secondary metabolites, both of which are linked to plant resistance. These genes should be further characterised before confirming their role in resistance to CBSD.     

Biochemical alterations in leaves of resistant and susceptible cotton genotypes infected systemically by cotton leaf curl Burewala virus

Leaf curl disease caused by Cotton Leaf Curl Burewala virus (CLCuBuV) has been recognized as serious threat to cotton in Indian subcontinent. However, information about cotton–CLCuBuV interaction is still limited. In this study, the level of phenolic compounds, total soluble proteins, and malondialdehyde (MDA) and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), proteases, superoxide dismutase (SOD), and polyphenol oxidase (PPO) were studied in leaves of two susceptible (CIM-496 & NIAB-111) and two resistant (Ravi and Co Tiep Khac) cotton genotypes. Disease symptoms were mild in the resistant genotypes but were severe in highly susceptible genotypes. The results showed that phenolic compounds, proteins, PAL, POX, CAT, proteases, SOD, PPO, and MDA play an active role in disease resistance against CLCuBuV. The amount of total phenols, proteases, MDA, and PPO was significantly higher in leaves of CLCuBuV-inoculated plants of both resistant genotypes as in non-inoculated plants, and decreased in CLCuBuV-inoculated plants of both susceptible genotypes over their healthy plants. POX, protein content, SOD, and PAL activities showed lower values in resistant genotypes, while they decreased significantly in susceptible genotypes as compared to the noninoculated plants except PAL, which showed non-significant decrease. CAT was found to be increased in both susceptible and resistant genotypes with maximum percent increase in resistant genotype Ravi, as compared to non-inoculated plants. The results showed significantly higher concentrations of total phenols and higher activity of protease, MDA, SOD, and PPO in resistant genotype Ravi after infection with CLCuBuV, suggesting that there is a correlation between constitutive induced levels of these enzymes and plant resistance that could be considered as biochemical markers for studying plant-virus compatible and incompatible interactions.     

The Relationship Between Peroxidase Activity and Resistance to Sphaerotheca fuliginea in Melons

The levels of peroxidase activity in leaves of non-infected melon plants resistant to Sphaerotheca fuliginea (Schl. ex. Fr.) Poll. were considerably higher than those in the leaves of susceptible plants. After infection the ratio of peroxidase activity to that in non-infected plants reached a value of up to 47.5 in susceptible plants whereas in resistant plants it remained between 1 and 2. Changes in isozyme pattern were investigated by polyacrylamide gel electrophoresis. In non-infected resistant leaves slowly migrating isozymes were found. Their intensities increased with time following infection. These isozymes were absent in the susceptible leaves but appeared after the leaves had been infected with S. fuliginea. The role of these isozymes in the resistance mechanism is discussed.     

Virus-induced silencing of <Emphasis Type="Italic">Comt</Emphasis>, p<Emphasis Type="Italic">Amt</Emphasis> and <Emphasis Type="Italic">Kas</Emphasis> genes results in a reduction of capsaicinoid accumulation in chili pepper fruits

Capsaicinoids are responsible for the pungent taste of chili pepper fruits of Capsicum species. Capsaicinoids are biosynthesized through both the phenylpropanoid and the branched-fatty acids pathways. Fragments of Comt (encoding a caffeic acid O-methyltransferase), pAmt (a putative aminotransferase), and Kas (a β-keto-acyl-[acyl-carrier-protein] synthase) genes, that are differentially expressed in placenta tissue of pungent chili pepper, were individually inserted into a Pepper huasteco yellow veins virus (PHYVV)-derived vector to determine, by virus-induced gene silencing, irrespective of whether these genes are involved in the biosynthesis of capsaicinoids. Reduction of the respective mRNA levels as well as the presence of related siRNAs confirmed the silencing of these three genes. Morphological alterations were evident in plants inoculated with PHYVV::Comt and PHYVV::Kas constructs; however, plants inoculated with PHYVV::pAmt showed no evident alterations. On the other hand, fruit setting was normal in all cases. Biochemical analysis of placenta tissues showed that, indeed, independent silencing of all three genes led to a dramatic reduction in capsaicinoid content in the fruits demonstrating the participation of these genes in capsaicinoid biosynthesis. Using this approach it was possible to generate non-pungent chili peppers at high efficiency.     

The adaptation of tomato mosaic virus to resistant tomato plants

Tomato mosaic virus derived from susceptible tomato plants (the standard virus) was cultured in resistant plants. Sap from non-inoculated leaves of resistant tomato plants infected with virus from the resistant host was more infective and contained more virus particles than leaf sap of resistant plants infected with the standard virus. Leaves of resistant tomatoes infected with virus from the resistant host also showed more obvious symptoms. Susceptible plants infected with virus from resistant plants not only showed fewer symptoms than when infected with standard virus, but samples were less infective and contained less virus up to 26 weeks, when values for infectivity were similar. This modification in activity was not reversible and was obtained with two lines of tomato having different types of resistance. Passage of virus from resistant plants through susceptible plants did not impair its ability to infect resistant plants.     

A developmentally regulated lipocalin-like gene is overexpressed in Tomato yellow leaf curl virus-resistant tomato plants upon virus inoculation, and its silencing abolishes resistance

To discover genes involved in tomato resistance to Tomato yellow leaf curl virus (TYLCV), we previously compared cDNA libraries from susceptible (S) and resistant (R) tomato lines. Among the genes preferentially expressed in R plants and upregulated by TYLCV infection was a gene encoding a lipocalin-like protein. This gene was termed Solanum lycopersicum virus resistant/susceptible lipocalin (SlVRSLip). The SlVRSLip structural gene sequence of R and S plants was identical. SlVRSLip was expressed in leaves during a 15-day window starting about 40?days after sowing (20?days after planting). SlVRSLip was upregulated by Bemisia tabaci (the TYLCV vector) feeding on R plant leaves, and even more strongly upregulated following whitefly-mediated TYLCV inoculation. Silencing of SlVRSLip in R plants led to the collapse of resistance upon TYLCV inoculation and to a necrotic response along the stem and petioles accompanied by ROS production. Contrary to previously identified tomato lipocalin gene DQ222981, SlVRSLip was not regulated by cold, nor was it regulated by heat or salt. The expression of SlVRSLip was inhibited in R plants in which the hexose transporter gene LeHT1 was silenced. In contrast, the expression of LeHT1 was not inhibited in SlVRSLip-silenced R plants. Hence, in the hierarchy of the gene network conferring TYLCV resistance, SlVRSLip is downstream of LeHT1. Silencing of another gene involved in resistance, a Permease-I like protein, did not affect the expression of SlVRSLip and LeHT1; expression of the Permease was not affected by silencing SlVRSLip or LeHT1, suggesting that it does not belong to the same network. The triple co-silencing of SlVRSLip, LeHT1 and Permease provoked an immediate cessation of growth of R plants upon infection and the accumulation of large amounts of virus. SlVRSLip is the first lipocalin-like gene shown to be involved in resistance to a plant virus.     

Blockage of stylet tips as the mechanism of resistance to virus transmission by Aphis gossypii in melon lines bearing the Vat gene

Aphis gossypii is the main virus vector in muskmelon crops. The melon gene Vat confers resistance to non‐persistent virus transmission by this aphid. The mechanism of this resistance is not well understood, but no relationship has been detected between resistance and the probing behaviour of aphids on resistant plants. Results presented here suggest that temporary blockage of aphid stylet tips preventing virus particle release may explain the resistance conferred by Vat gene. We performed experiments in which viruliferous aphids were allowed to probe different sequences of resistant (Vat‐bearing) and/or susceptible melon plants. The results demonstrated that A. gossypii inoculates Cucumber mosaic virus (CMV) efficiently in susceptible plants having previously probed resistant plants, showing that the resistance mechanism is reversible. Furthermore, the infection rate obtained for susceptible plants was the same (25%) regardless of whether the transmitting aphid had come directly from the CMV source or had subsequently probed on resistant plants. This result suggests that virus is not lost from stylet to plant during probing of resistant plants, supporting the temporary blockage hypothesis. We also found that the ability of Myzus persicae to transmit CMV is noticeably reduced after probing on resistant plants, providing evidence that this aphid species also responds to the presence of the Vat gene. Finally, we also found that in probes immediately after virus acquisition M. persicae inoculates resistant plants with CMV more efficiently than susceptible plants, perhaps because the Vat gene product induces increased salivation by this aphid.     

Screening for Barley yellow dwarf virus-Resistant Barley Genotypes by Assessment of Virus Content in Inoculated Seedlings

The content of Barley yellow dwarf virus (BYDV) in roots and leaves of barley seedling plants differing in their level of resistance was assessed by quantitative ELISA 1–42 days after inoculation with the strain of BYDV (PAV). High virus accumulation in roots and low concentration in leaves was characteristic of the period 9–15 days after inoculation. In leaves, the differences in virus content between resistant and susceptible genotypes became significant after 15 days and resistance to virus accumulation was better expressed 30–39 days after inoculation. Roots of resistant materials exhibited evident retardation of virus accumulation and the greatest difference in virus content between resistant and susceptible plants was detected 9 days after inoculation. By these criteria, the selected winter and spring barley cultivars and lines (in total 44 materials) fell in to five groups according to field reactions and the presence or absence of the Yd2 resistance gene. There were highly significant and positive relations between ELISA values and 5‐year field data on symptomatic reactions and grain‐yield reductions due to infection. Using the described method, resistant and moderately resistant genotypes (both Yd2 and non‐Yd2) were significantly differentiated from susceptible genotypes. The possible use of this method in screening for BYDV resistance is discussed.     

Evidence that RNA silencing-mediated resistance to beet necrotic yellow vein virus is less effective in roots than in leaves

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.     

花魔芋抗软腐病植株的鉴定及其抗性机理的初步研究

通过筛选获得了对魔芋(Amorphophallus konjac)软腐病具有较强抗性的花魔芋抗病植株, 经核型分析发现, 其染色体数目与普通植株一致, 均为2n=26。抗病实验结果表明, 该抗病植株对魔芋软腐病的抗性较普通植株强。采用酶联免疫吸附法, 测定了软腐病病原菌接种30小时内的抗病与普通植株叶片中内源植物激素(SA与JA)的含量变化。结果显示, 抗病和普通植株叶片中SA和JA含量变化的总体趋势明显不同。主要表现在, 抗病和普通植株叶片中SA和JA积累的时间不同; 此外, 积累的量也有明显差异。推测魔芋抗病植株可能存在与目前大多数植株不同的抗病机制。     

Revealing the importance of meristems and roots for the development of hypersensitive responses and full foliar resistance to Phytophthora infestans in the resistant potato cultivar Sarpo Mira

The defence responses of potato against Phytophthora infestans were studied using the highly resistant Sarpo Mira cultivar. The effects of plant integrity, meristems, and roots on the hypersensitive response (HR), plant resistance, and the regulation of PR genes were analysed. Sarpo Mira shoots and roots grafted with the susceptible Bintje cultivar as well as non-grafted different parts of Sarpo Mira plants were inoculated with P. infestans. The progress of the infection and the number of HR lesions were monitored, and the regulation of PR genes was compared in detached and attached leaves. Additionally, the antimicrobial activity of plant extracts was assessed. The presented data show that roots are needed to achieve full pathogen resistance, that the removal of meristems in detached leaves inhibits the formation of HR lesions, that PR genes are differentially regulated in detached leaves compared with leaves of whole plants, and that antimicrobial compounds accumulate in leaves and roots of Sarpo Mira plants challenged with P. infestans. While meristems are necessary for the formation of HR lesions, the roots of Sarpo Mira plants participate in the production of defence-associated compounds that increase systemic resistance. Based on the literature and on the presented results, a model is proposed for mechanisms involved in Sarpo Mira resistance that may apply to other resistant potato cultivars.     

The impact of Meu1-mediated resistance in tomato on longevity, fecundity and behavior of the potato aphid, Macrosiphum euphorbiae

The effect of the tomato resistance gene, Meu1, on feeding, longevity, fecundity and developmental rate of the pink biotype of the potato aphid, Macrosiphum euphorbiae (Thomas) (Hemiptera, Aphididae), was determined using nearly isogenic tomato (Lycopersicon esculentum Mill, Solanaceae) lines. Aphid mortality was significantly higher on resistant plants, with 60% of the aphids dying by the 4th day of exposure. By the 10th day, all the aphids on the resistant plants were dead whereas 100% of the aphids on susceptible plants were alive. Meu1-mediated resistance resulted in significantly decreased fecundity with a ten-fold decrease in the net fertility rate (4.5 and 45.7 progeny per aphid on resistant and susceptible tomato, respectively). A qualitative analysis showed that honeydew was produced by aphids on resistant and susceptible plants, suggesting that aphids initiate feeding on both plant types. However, significantly lower quantities of honeydew were present when aphids were caged on resistant plants. There were also significant differences in aphid location on resistant and susceptible leaves. Experiments evaluating behavior in less than 24 h showed that aphids left resistant leaves after relatively short exposure (3–6 h). Aphids transferred from resistant to susceptible tomato at intervals between 3 h and 24 h resumed feeding as evidenced by presence of honeydew. Although the mechanism by which Meu1-mediated resistance operates is not yet known, our data suggest that resistance factors act rapidly after initiation of feeding and that lower fecundity and longevity are related to reduction in aphid feeding.     

Characteristics of resistance in rice to rice hoja blanca virus (RHBV) and its vector, Tagosodes orizicolus (Muir)

This paper characterises resistance to rice hoja blanca virus (RHBV) which is transmitted by the planthopper Tagosodes orizicolus (Muir). Resistance is expressed as decreased proportion of plants infected compared to susceptible lines, although severity of symptom expression is similar in both types. This resistance is not due to differences in vector feeding behaviour. Vectors reared eight generations on resistant plants showed no increased ability to transmit to resistant lines or decreased ability to transmit to susceptible ones. Longevity of vectors was similar when reared either on virus-resistant or susceptible plants. Incubation period of the virus in resistant plants are significantly longer than in susceptible plants. Resistance increased with plant age in resistant and susceptible cultivars. Increased virus dosage, as determined by increased number of viru-liferous vectors per inoculated plant, caused an increase in transmission to both resistant and susceptible cultivars. However, the ranking of resistant and susceptible remained the same across the experimental range of dosage and plant age. It is concluded that the resistance studied is to virus infection and there is little risk of “breakdown” occurring as a result of genetic or behavioural changes in the vector population. This will permit the use of economic thresholds to planthopper feeding damage with little risk of RHBV epidemic outbreak.