Molecular Plant Microbe Interactions

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced th...

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype. ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC¯ background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC¯ strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.

Genetic complementation of a spontaneous mutant, impaired in flocculation, Congo red binding, and colonization of root surface, led to the identification of a new regulatory gene in Azospirillum brasilense Sp7, designated flcA. The deduced amino acid sequence of flcA shared high similarity with a family of transcriptional activators of the LuxR-UhpA family. The most significant match was with the AgmR protein, an activator for glycerol metabolism in Pseudomonas aeruginosa. Derivatives of Sp7 resulting from site-directed Tn5 mutagenesis in the flcA coding sequence were constructed by marker exchange. Characterization of the resulting mutant strains showed that flcA controls the production of capsular polysaccharides, the flocculation process in culture, and the colonization of the root surface of wheat. This study provides new information on the genetic control of the mechanism of plant root colonization by Azospirillum.

In this work the bio-availability of amino acids for the root-colonizing Pseudomonas fluorescens strain WCS365 in the tomato rhizosphere was studied. The amino acid composition of axenically collected tomato root exudate was determined. The results show that aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino acid components. The concentrations of individual amino acids in the rhizosphere of gnotobiotically grown tomato plants were estimated and considered to be too low to support growth of rhizosphere micro-organisms to numbers usually found in the tomato rhizosphere. To test this experimentally, mutants of P. fluorescens WCS365 auxotrophic for the amino acids leucine, arginine, histidine, isoleucine plus valine, and tryptophan were isolated after mutagenesis with Tn5lacZ. Root tip colonization of these mutants was measured after inoculation of germinated tomato seeds and subsequent growth in a gnotobiotic quartz sand system (M. Simons, A. J. van der Bij, I. Brand, L. A. de Weger, C. A. Wijffelman, and B. J. J. Lugtenberg. 1996. Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 9:600-607). In contrast to the wild-type strain, none of the five amino acid auxotrophs tested was able to colonize the tomato root tip, neither alone nor after co-inoculation with the wild-type strain. However, addition of the appropriate amino acid to the system restored colonization by the auxotrophic mutants, usually to wild-type levels. Analysis of the root base showed that cells of auxotrophic mutants were still present there. The results show that, although amino acids are present in root exudate, the bio-availability of the tested amino acids is too low to support root tip colonization by auxotrophic mutants of P. fluorescens strain WCS365. The genes that are required for amino acid synthesis are therefore necessary for root colonization. Moreover, these compounds apparently play no major role as nutrients in the tomato rhizosphere.

Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobiainduced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars ofXanthomonas campestris species. The 16 strains ofX. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.

ISSN: 2319-7706 Volume 8 Number 11 (2019) Journal homepage: http://www.ijcmas.com Turcicum leaf blight of maize caused by Exserohilum turcicum is an economically important foliar disease, the findings of the study reveals that, among the 32 isolates, tested for mycelia growth on PDA, Aedigudda (Et12), Dibbadahalli (Et14), Hulihyder (Et19), Kanakagiri (Et28) and Raichur (Et32) isolate showed an excellent mean radial growth (90.00 mm) and least mean radial growth was obtained in Et9 (Choornoor) isolate with mycelia growth of 69.00 mm. Maximum number of E. turcicum isolates were having the septa of 3-8 and 3-9 with protruding hilum. Interestingly an isolate Et16 and Et28 from Hadagali and Kanakagiri respectively having 3-15 septa. Maximum size of conidia was observed in Et16 of Hadagali (120.78 μm × 29.15 μm) followed by Et28 of Kanakagiri (116.62 μm × 24.99 μm). In pathogen variability studies, CM-202 and MI-39 inbred lines showed significantly higher mean lesion sizes of 1.31 cm and ...

One of the major issues facing humankind is global food security. A changing climate, coupled with a heightened consumer awareness of how food is produced and legislative changes governing the usage of agrichemicals for improving plant health and yield, means that alternative, more integrated and sustainable approaches are needed for crop management practices. To this end, there is increasing recognition of the value of the role of microbial inoculants in agriculture. The focus of this review is to understand how plant-growth-promoting bacteria and arbuscular mycorrhizal fungi can play a part in improving crop yield by promoting the health status of the plant through the sequestration of various nutrients and in the control of plant diseases.

Global food security is vulnerable due to massive growth of the human population, changes in global climate, the emergence of novel/more virulent pathogens, and demands from increasingly discerning consumers for chemical-free, sustainably produced food products. Bacterium- based biological control agents (BCAs), if used as part of an integrated management system, may satisfy the above demands. We focus on the advantages, limitations, problems, and challenges involved in such strategies.

Identification of host proteins interacting with the aphid-borne, Potato leafroll virus (PLRV) from the genus Polerovirus, family Luteoviridae, is a critical step towards understanding how PLRV and related viruses infect plants. However, the tight spatial distribution of PLRV to phloem tissues poses challenges. A polyclonal antibody raised against purified PLRV virions was used to co-immunoprecipitate virus-host protein complexes from Nicotiana benthamiana tissue inoculated with an infectious PLRV cDNA clone using Agrobacterium tumefaciens. A. tumefaciens-mediated delivery of PLRV enabled infection and production of assembled, insect-transmissible virus in most leaf cells overcoming the dynamic range constraint posed by a systemically infected host. Isolated protein complexes were characterized using high-resolution mass spectrometry and were comprised of host proteins interacting directly and/or indirectly with virions, as well as the non-incorporated readthrough protein (RTP) and ...

In our paper we challenge the common notion that small and medium sized enterprises (SMEs) have to adapt to the international business environment to be successful when acting in an international context. Although we believe in a need for certain adaption, we claim that the possibility that SME are to a certain extent able to proactively shape outer conditions is not adequately considered in literature. Therefore we ask how far SMEs in international business need to adapt to the environment to ensure a state of international competitiveness. We understand SMEs as open systems (Sanchez and Heene, 1996) and employ the Competence-based Theory of the Firm (CbTF) (Foss and Ishikawa, 2007; Freiling et al., 2008) as theoretical frame of reference for our conceptual paper. Thanks to its moderate voluntaristic nature, this theory allows considering both social embeddedness and managerial discretion of firms. Besides, this perspective takes into account internal developments as well as developments in the business environment. This is particularly useful to understand interaction processes with the environment that aim at accessing so-called 'firm-addressable assets'. Firm-addressable assets explain the resource dependence of international SMEs on third-parties. However, backed by 'absorptive capacity' (Cohen and Levinthal, 1990), firm-addressable assets fuel internal processes of learning as well as resource and competence building. Moreover, CbTF allows for considering the role of certain categories of resources. Following service-dominant logic (SDL) (Vargo and Lusch, 2004), the distinction between 'operand resources' and 'operant resources' becomes a centerpiece of our argument: SMEs need to adapt to the environment due to resource constraints by operand resources and shape the environment primarily driven by operant resources. Our paper advances research in three ways: (1) we reinforce the concept of moderate voluntarism in this stream of research and challenge the predominantly deterministic view on SME environmental adaption; (2) we introduce a rather new theory (CbTF) and develop an understanding that features the managerial discretion of SME's in international competition; (3) we depart from the quite unspecific notion that intangibles matter in case of proactive SME management and specify the resource categories in this realm with more explanatory power in this regard.

Worldwide, contamination of soil and ground water is a severe problem. The negative effects of pollutants on the environment and on human health are diverse and depend on the nature of the pollution. The search for alternative methods for excavation and incineration to clean polluted sites resulted in the application of bioremediation techniques. In this review, we describe some generally accepted bioremediation tools and subsequently focus on the combination of two approaches, phytoremediation and bioaugmentation, resulting in rhizoremediation. During rhizoremediation, exudates derived from the plant can help to stimulate the survival and action of bacteria, which subsequently results in a more efficient degradation of pollutants. The root system of plants can help to spread bacteria through soil and help to penetrate otherwise impermeable soil layers. The inoculation of pollutant-degrading bacteria on plant seed can be an important additive to improve the efficiency of phytoremediation or bioaugmentation.

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1-3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear . Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature and consequent ability to chelate divalent calcium ions . In plants, Ca 2+ ion influx to the cytosol from the apoplast (where bacteria multiply ) is a prerequisite for

Oxylipins, a class of oxygenase-derived unsaturated fatty acids, are important signal molecules in many biological systems. Recent characterization of an Aspergillus flavus lipoxygenase gene, lox, revealed its importance in maintaining a density-dependent morphology switch from sclerotia to conidia as population density increased. Here, we present evidence for the involvement of four more oxylipingenerating dioxygenases (PpoA, PpoB, PpoC, and PpoD) in A. flavus density-dependent phenomena and the effects of loss of these genes on aflatoxin production and seed colonization. Although several single mutants showed alterations in the sclerotia-to-conidia switch, the major effect was observed in a strain downregulated for all five oxygenases (invert repeat transgene [IRT] strain IRT4 = ppoA, ppoB, ppoC, ppoD, and lox). In strain IRT4, sclerotia production was increased up to 500-fold whereas conidiation was decreased down to 100-fold and the strain was unable to switch into conidial production. Aflatoxin (AF) production for all mutant strains and the wild type was greatest at low population densities and absent in high populations except for strain IRT4, which consistently produced high levels of the mycotoxin. Growth on host seed by both IRT4 and IRT2 (downregulated in ppoA, ppoB, and ppoD) was marked by decreased conidial but increased AF production. We propose that A. flavus oxygenases and the oxylipins they produce act in a highly interdependent network with some redundancy of biological function. These studies provide substantial evidence for oxylipin-based mechanisms in governing fungus-seed interactions and in regulating a coordinated quorum-sensing mechanism in A. flavus.

Plant pathogen attacks are perceived through pathogenissued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models.

Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Additionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host-pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virusinduced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions currently used by the research community. In addition to addressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.

A strategy to control the devastating late blight disease is providing potato cultivars with genes that are effective in resistance to a broad spectrum of Phytophthora infestans isolates. Thus far, most late blight resistance (R) genes that were introgressed in potato were quickly defeated. In contrast, the Rpi-blb1 gene originating from Solanum bulbocastanum has performed as an exclusive broad-spectrum R gene for many years. Recently, the RXLR effector family ipiO was identified to contain Avr-blb1. Monitoring the genetic diversity of the ipiO family in a large set of isolates of

Considering the complexity of the in vivo interactions established by a mycoparasitic biocontrol agent at the plant rhizosphere, proteomic, genomic, and transcriptomic approaches were used to study a novel Trichoderma gene coding for a plant cell wall (PCW)-degrading enzyme. A proteome analysis, using a three-component (Trichoderma spp.-tomato plantlets-pathogen) system, allowed us to identify a differentially expressed Trichoderma harzianum endopolygalacturonase (endoPG). Spot 0303 remarkably increased only in the presence of the soilborne pathogens Rhizoctonia solani and Pythium ultimum, and corresponded to an expressed sequence tag from a T. harzianum T34 cDNA library that was constructed in the presence of PCW polymers and used to isolate the Thpg1 gene. Compared with the wild-type strain, Thpg1-silenced transformants showed lower PG activity, less growth on pectin medium, and reduced capability to colonize tomato roots. These results were combined with microarray comparative data from the transcriptome of Arabidopsis plants inoculated with the wild type or a Thpg1-silenced transformant (ePG5). The endoPG-encoding gene was found to be required for active root colonization and plant defense induction by T. harzianum T34. In vivo assays showed that Botrytis cinerea leaf necrotic lesions were slightly smaller in plants colonized by ePG5, although no statistically significant differences were observed.

RNA silencing is a conserved eukaryotic process mediated by small RNA molecules that inhibit gene expression at the transcriptional, mRNA-stability or translational level through sequence-specific interactions. Diverse roles have been identified for RNA silencing such as genome defence against mobile DNA elements or downregulation of specific factors during plant and animal development. In plants, RNA silencing plays a crucial role in antiviral defence by inhibiting viral accumulation and sometimes preventing systemic infection. As a counter-defence mechanism, viruses have evolved a set of anti-silencing strategies, of which the most common is the production of viral suppressors of RNA silencing (VSRs). Here we review the different strategies underlying VSRs action including prevention of viral-derived small (vs)RNAs synthesis, vsRNAs sequestration or inhibition of vsRNA-guided effector complexes. We will also underline the consequences of this molecular arms race on the evolution of both viral and host genomes.

Numerous salmonellosis outbreaks have been associated with vegetables, in particular sprouted seed. Thin aggregative fimbriae (Tafi), a component of the extracellular matrix responsible for multicellular behavior, are important for Salmonella enterica attachment and colonization of plants.

Sclerotinia sclerotiorum is a necrotrophic, omnivorous plant pathogen with worldwide distribution. Sclerotia of S. sclerotiorum are pigmented, multihyphal structures that play a central role in the life and infection cycles of this pathogen. Calcineurin, a Ser/Thr phosphatase linked to several signal-transduction pathways, plays a key role in the regulation of cation homeostasis, morphogenesis, cellwall integrity, and pathogenesis in fungi. We demonstrate that calcineurin expression in S. sclerotiorum is altered in a phase-specific manner during sclerotial development. Inhibition of calcineurin by FK506, cysclosporin A, or inducible antisense calcineurin expression impaired sclerotial development at the prematuration phase and increased germination of preformed sclerotia. Induction of antisense calcineurin expression in S. sclerotiorum resulted in reduced pathogenesis on tomato and Arabidopsis. However, secretion of oxalic acid, a key virulence factor of S. sclerotiorum, was not altered. Inhibition of calcineurin conferred a reduction in cell wall β-1,3-glucan content and increased sensitivity to cell-wall-degrading enzymes and to the glucan synthase inhibitor caspofungin. Thus, calcineurin plays a major role in both sclerotial development and pathogenesis of S. sclerotiorum and, most likely, other phytopathogens.

Pseudomonas aeruginosa 7NSK2 induces resistance in dicots through a synergistic interaction of the phenazine pyocyanin and the salicylic acid-derivative pyochelin. Root inoculation of the monocot model rice with 7NSK2 partially protected leaves against blast disease (Magnaporthe grisea) but failed to consistently reduce sheath blight (Rhizoctonia solani). Only mutations interfering with pyocyanin production led to a significant decrease in induced systemic resistance (ISR) to M. grisea, and in trans complementation for pyocyanin production restored the ability to elicit ISR. Intriguingly, pyocyanin-deficient mutants, unlike the wild type, triggered ISR against R. solani. Hence, bacterial pyocyanin plays a differential role in 7NSK2-mediated ISR in rice. Application of purified pyocyanin to hydroponically grown rice seedlings increased H202 levels locally on the root surface as well as a biphasic H202 generation pattern in distal leaves. Co-application of pyocyanin and the antioxidant sodium ascorbate alleviated the opposite effects of pyocyanin on rice blast and sheath blight development, suggesting that the differential effectiveness of pyocyanin with respect to 7NSK2-triggered ISR is mediated by transiently elevated H202 levels in planta. The cumulative results suggest that reactive oxygen species act as a double-edged sword in the interaction of rice with the hemibiotroph M. grisea and the necrotroph R. solani.

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glyc...

Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for 'PR37Y15' but not 'DK315', as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.

Phytoplasmas are cell wall-less bacteria inhabiting the phloem and utilizing it for their spread. Infected plants often show changes in growth pattern and a reduced crop yield. A quantitative real-time polymerase chain reaction (Q-PCR) assay and a bioimaging method were developed to quantify and localize phytoplasmas in situ. According to the Q-PCR assay, phytoplasmas accumulated disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser extent, in petioles of source leaves and in stems. However, phytoplasma accumulation was small or nondetectable in sink organs (roots and sink leaves). For bioimaging, infected plant tissue was stained with vital fluorescence dyes and examined using confocal laser scanning microscopy. With a DNA-sensitive dye, the pathogens were detected exclusively in the phloem, where they formed dense masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by counterstaining with aniline blue for callose and multiphoton excitation. With a potentiometric dye, not all DNA-positive material was stained, suggesting that the dye stained metabolically active phytoplasmas only. Some highly infected sieve tubes contained phytoplasmas that were either inactive or dead upon staining.

Systemically induced resistance is a promising strategy to control plant diseases, as it affects numerous pathogens. However, since induced resistance reduces one or both growth and activity of plant pathogens, the indigenous microflora may also be affected by an enhanced defensive state of the plant. The aim of this study was to elucidate how much the bacterial rhizosphere microflora of Arabidopsis is affected by induced systemic resistance (ISR) or systemic acquired resistance (SAR). Therefore, the bacterial microflora of wild-type plants and plants affected in their defense signaling was compared. Additionally, ISR was induced by application of methyl jasmonate and SAR by treatment with salicylic acid or benzothiadiazole. As a comparative model, we also used wild type and ethylene-insensitive tobacco. Some of the Arabidopsis genotypes affected in defense signaling showed altered numbers of culturable bacteria in their rhizospheres; however, effects were dependent on soil type. Effects of plant genotype on rhizosphere bacterial community structure could not be related to plant defense because chemical activation of ISR or SAR had no significant effects on density and structure of the rhizosphere bacterial community. These findings support the notion that control of plant diseases by elicitation of systemic resistance will not significantly affect the resident soil bacterial microflora.

Plant viruses are disseminated by either vertical (vegetative multiplication or sexual reproduction) or horizontal (vector-mediated) propagation. Plant pararetrovirusesmembers of the Caulimoviridae family-have developed an alternative strategy for vertical propagation via integration within the host plant genome, although integration is not required for viral replication. Integrated endogenous pararetrovirus (EPRV) sequences have undergone extensive viral genome rearrangements and contain more than one copy of the viral genome. Furthermore, EPRV can become infectious upon spontaneous escape of active virus following stresses such as wounding, tissue culture, or interspecific crosses. Such infectious EPRV are of great importance, not only in terms of their ability to precipitate epidemic outbreaks but also because of their effect on breeding of numerous plant genomes in temperate and tropical crops. This is especially true for banana, a crop susceptible to banana streak viruses, the causative agents of banana streak disease. Thus, the classical three-component banana-Banana streak virus (BSV)-mealybug pathosystem can be expanded to include endogenous BSV as an alternative source of active virions. The BSV-banana pathosystem is one of only three pathosystems known to date to harbor this remarkable feature, and the present review focuses exclusively on it to illustrate this four-partner interaction.

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called ω that is important for virulence. Previous results from our laboratory indicated that the Cterminal, bipartite NLS and the ω region are not essential for nuclear uptake of T-DNA, and further suggested that the ω domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the ω domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of β-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl β-Dglucuronic acid (X-gluc). However, using an ω-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 ω deletion/substitution. Taken together, these data indicate that the VirD2 ω domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the ω mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an ω mutation. In addition, a mutant VirD2 protein lacking the ω domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino ac-

First of all I would like to thank the Almighty God for giving me the aptitude, endurance, determination and guidance throughout the ups and downs of life. With your light, I saw my way! Several people have assisted me during my research work. Although it is not possible to mention all in a few sentences I would like to thank those who have been particularly important to my work. I feel great pleasure to express my special thanks to my supervisor Dr. Justus Wesseler for his critical and valuable comments in the course of this study. His insightful comments for the betterment of the whole work were appreciable. I would like to express my deepest gratitude to my external supervisor Dr. Huib Hengsdijk from Plant Research International for his invaluable insightful comments, guidance and assistance in finding funding for my research. With out unlimited support and guidance of my supervisors throughout the research work, this thesis would not be in this format. My particular gratitude goes to the Horn of Africa Regional Environmental Network and Centre for its financial support. I would like to thank Dr. Satishkumar Belliethathan and Mr. Bart Jan van Beuzekom from Horn of African Regional Environmental Network and Centre for their generous cooperation and facilitations of financial issues. My special thank go to Werer Agricultural Research Centre manager, Mr. Taddele Amde, for arranging transport facilities for my research. Transaction costs, i.e. time for negotiating contracts, motivating the members and costs for coordination and monitoring of scheme activities were higher in Dodicha due to poor scheme management. Environmental problems associated with the small-scale irrigation schemes were assessed only qualitatively. Farmers argue that water is used efficiently since pumping bears fuel costs. Further research should investigate this claim also in relation to the relatively low crop yields obtained in both schemes. To improve the economic and environmental performance of small scale-irrigation schemes institutional support (input supply, output marketing and credit services), training of farmers on improved crop and water management issues, regular supervision and monitoring of scheme activities are crucial.

The hrp/wts gene cluster of Pantoea stewartii subsp. stew-artii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide se-quencing were used to identify hrp/wts genes within the ...

Elicitins form a family of structurally related proteins that induce the hypersensitive response (HR) in plants, particularly Nicotiana spp. The elicitin family is composed of several classes. Most species of the plant-pathogenic oomycete genus Phytophthora produce the well-characterized 10-kDa canonical elicitins (class I), such as INF1 of the potato and tomato pathogen Phytophthora infestans. Two genes, inf2A and inf2B, encoding a distinct class (class III) of elicitinlike proteins, also occur in P. infestans. Unlike secreted class I elicitins, class III elicitins are thought to be cell-surface-anchored polypeptides. Molecular characterization of the inf2 genes indicated that they are widespread in Phytophthora spp. and occur as a small gene family. In addition, Southern blot and Northern blot hybridizations using gene-specific probes showed that inf2A and inf2B genes and transcripts can be detected in 17 different P. infestans isolates. Functional secreted expression in plant cells of the elicitin domain of the inf1 and inf2 genes was conducted using a binary Potato virus X (PVX) vector (agroinfection) and Agrobacterium tumefaciens transient transformation assays (agroinfiltration), and resulted in HR-like necrotic symptoms and induction of defense response genes in tobacco. However, comparative analyses of elicitor activity of INF1, INF2A, and INF2B revealed significant differences in intensity, specificity, and consistency of HR induction. Whereas INF1 induced the HR in Nicotiana benthamiana, INF2A induced weak symptoms and INF2B induced no symptoms on this plant. Nonetheless, similar to INF1, HR induction by INF2A in benthamiana required the ubiquitin ligase-associated protein SGT1. Overall, these results suggest that variation in the resistance of Nicotiana spp. to P. infestans is shadowed by variation in the response to INF elicitins. The ability of tobacco, but not N. benthamiana, to respond to INF2B could explain differences in resistance to P. infestans observed for these two species.

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F. oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.

Erwinia amylovora is a gram-negative phytopathogen that causes fire blight of pome fruit and related members of the family Rosaceae. We sequenced the putative autoinducer-2 (AI-2) synthase gene luxS from E. amylovora. Diversity analysis indicated that this gene is extremely conserved among E. amylovora strains. Quorum sensing mediated by LuxS has been implicated in coordinated gene expression, growth, and virulence in other enterobacteria; however, our evidence suggests this is not the function in E. amylovora. Mutational analysis pointed to a role in colonization of apple blossoms, the primary infection court for fire blight, although little if any role in virulence on apple shoots and pear fruit was observed. Expression of key virulence genes hrpL and dspA/E was reduced in mutants of two E. amylovora strains. Stronger effects on gene expression were observed for metabolic genes involved in the activated methyl cycle with mutants having greater levels of expression. No quorum-sensing effect was observed in coculture experiments with wild-type and mutant strains either in vitro or in apple blossoms. Known receptors essential for AI-2 quorum sensing, the LuxPQ sensor kinase or the Lsr ABC-transporter, are absent in E. amylovora, further suggesting a primarily metabolic role for luxS in this bacterium.

Plant resistance to pathogens is commonly associated with a hypersensitive response (HR), but the degree to which the HR is responsible for incompatibility is subject to debate. Resistance to aphids is likely to share features with resistance to pathogens but is less well understood. Here, we report effective resistance to the pea aphid Acyrthosiphon pisum in Medicago truncatula. Aphids lost weight and died rapidly (within two days) on the resistant genotype Jemalong, which developed necrotic lesions following infestation. Lesions were induced by nonvascular intracellular stylet punctures by aphids, remained localized to the site of stylet entry, stained for the presence of reactive oxygen species, and were similar to the HR induced by the bacterial pathogen Pseudomonas syringae pv. phaseolicola. The implication that aphid-induced lesions confer resistance was tested by quantitative trait loci analysis using recombinant inbred lines derived from a cross between Jemalong and the susceptible genotype DZA315.16. One major locus, RAP1, was identified that was sufficient to confer race-specific resistance against the pea aphid and was mapped to the middle of chromosome 3. Surprisingly, a separate locus, mapping to the top of chromosome 3, governed aphidinduced HR, indicating that the HR-like lesions are not required for RAP1-mediated aphid resistance.

The Tsw gene conferring dominant resistance to the Tospovirus Tomato spotted wilt virus (TSWV) in Capsicum spp. has been tagged with a random amplified polymorphic DNA marker and mapped to the distal portion of chromosome 10. No mapped homologues of Sw-5, a phenotypically similar dominant TSWV resistance gene in tomato, map to this region in C. annuum, although a number of Sw-5 homologues are found at corresponding positions in pepper and tomato. The relationship between Tsw and Sw-5 was also examined through genetic studies of TSWV. The capacity of TSWV-A to overcome the Tsw gene in pepper and the Sw-5 gene in tomato maps to different TSWV genome segments. Therefore, despite phenotypic and genetic similarities of resistance in tomato and pepper, we infer that distinct viral gene products control the outcome of infection in plants carrying Sw-5 and Tsw, and that these loci do not appear to share a recent common evolutionary ancestor.

A proportion of the Nod factors of some Rhizobium leguminosarum bv. trifolii strains is characterized by the presence of highly unsaturated fatty acyl chains containing trans double bonds in conjugation with the carbonyl group of the glycan oligosaccharide backbone. These fatty acyl chains are C18:3, C20:3, C18:4, or C20:4 and have UV-absorption maxima at 303 and 330 nm. These Nod factors are presumed to be important for host-specific nodulation on clover species. However, in wild-type R. leguminosarum bv. trifolii ANU843, Nod factors with these characteristic acyl chains were not observed using standard growth conditions. They were observed only when nod genes were present in multiple copies or when transcription was artificially increased to higher levels by introduction of extra copies of the transcriptional regulator gene nodD. In a screen for the genetic requirements for production of the Nod factors with these characteristic structures, it was found that the region downstream of nodF and nodE is essential for the presence of highly unsaturated fatty acyl moieties. Mu-lacZ insertion in this region produced a mutant that did not produce detectable levels of the highly unsaturated fatty acyl-bearing Nod factors. The Mu-lacZ insertion was translationally fused to a putative new gene, designated nodR, in the nodE-nodL intergenic region; however, no predicted function for the putative NodR protein has been obtained from database homology searches. In a set of 12 wild-type strains of R. leguminosarum by. trifolii originating from various geographical regions that were analyzed for the presence of a nodR-like gene, it was found that seven strains carry a homologous NodR open reading frame. Taken together, our results suggest a tightly controlled regulation of nod genes, in which we propose that it is the balance of transcriptional levels of nodFE and the nodRL genes that is critical for determining the presence of highly unsaturated fatty acyl moieties in the Nod factors produced by R. leguminosarum bv. trifolii.