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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.

Rhizobacteria–induced systemic resistance (ISR) and pathogen-induced systemic acquired resistance (SAR) have a broad, yet partly distinct, range of effectiveness against pathogenic microorganisms. Here, we investigated the effectiveness of ISR and SAR in Arabidopsis against the tissue-chewing insects Pieris rapae and Spodoptera exigua. Resistance against insects consists of direct defense, such as the production of toxins and feeding deterrents and indirect defense such as the production of plant volatiles that attract carnivorous enemies of the herbivores. Wind-tunnel experiments revealed that ISR and SAR did not affect herbivore-induced attraction of the parasitic wasp Cotesia rubecula (indirect defense). By contrast, ISR and SAR significantly reduced growth and development of the generalist herbivore S. exigua, although not that of the specialist P. rapae. This enhanced direct defense against S. exigua was associated with potentiated expression of the defense-related genes PDF1.2 and HEL. Expression profiling using a dedicated cDNA microarray revealed four additional, differentially primed genes in microbially induced S. exigua-challenged plants, three of which encode a lipid-transfer protein. Together, these results indicate that microbially induced plants are differentially primed for enhanced insect-responsive gene expression that is associated with increased direct defense against the generalist S. exigua but not against the specialist P. rapae.

Ran, L. X., van Loon, L. C., and Bakker, P. A. H. M. 2005. No role for bacterially produced salicylic acid in rhizobacterial induction of systemic resistance in Arabidopsis. Phytopathology 95:1349-1355. The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell –1 for WCS417r to >25 fg cell –1 for WCS374r. Addition of 200 µM FeCl3 to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28°C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36°C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis-related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.

The ability of selected strains of fluorescent Pseudomonas spp. to cause induced systemic resistance (ISR) in Eucalyptus urophylla against bacterial wilt caused by Ralstonia solanacearum was investigated. Four of the five strains used can produce salicylic acid (SA) in vitro and, therefore, chemical SA, that is known to induce resistance in many plant species, was used as a reference treatment. Whereas a soil drench with SA did induce systemic resistance in E. urophylla, infiltration of SA into leaves did not. None of the fluorescent Pseudomonas spp. strains caused ISR against bacterial wilt when applied to the soil, but two strains, P. putida WCS358r and P. fluorescens WCS374r triggered ISR when infiltrated into two lower leaves 3–7 days before challenge inoculation. A mutant of strain WCS358r defective in the biosynthesis of the fluorescent siderophore pseudobactin, did not cause ISR, while the purified siderophore of WCS358r did, suggesting that pseudobactin358 is the ISR determinant of WCS358. A siderophore-minus mutant of WCS374r induced the same level of disease resistance as its parental strain, but the purified siderophore induced resistance as well, indicating that both the siderophore and another, unknown, inducing determinant(s) of WCS374r can trigger ISR in Eucalyptus. A possible role of WCS374r-produced SA remains uncertain. Transformation of a siderophore-minus mutant of WCS358 with the SA biosynthetic gene cluster from WCS374 did not enable this transformant to cause ISR in E. urophylla.

Transgenic tobacco plants (Tetr) expressing the mutant etr1-1 gene from Arabidopsis thaliana are insensitive to ethylene and develop symptoms of wilting and stem rot when grown in nonautoclaved soil. Several isolates of Fusarium, Thielaviopsis, and Pythium were recovered from stems of diseased Tetr plants. Inoculation with each of these isolates of 6-week-old plants growing in autoclaved soil caused disease in Tetr plants but not in nontransformed plants. Also, when 2-week-old seedlings were used, nontransformed tobacco appeared nonsusceptible to the Fusarium isolates, whereas Tetr seedlings did develop disease. Tetr seedlings were not susceptible to several nonhost Fusarium isolates. In contrast to results with Fusarium isolates, inoculation of 2-week-old seedlings with a Thielaviopsis isolate resulted in equal symptom development of nontransformed and Tetr tobacco. In order to explore the potential range of pathogens to which Tetr tobacco plants display enhanced susceptibility, the pathogenicity of several root and leaf pathogens was tested. Tetr plants were more susceptible to the necrotrophic fungi Botrytis cinerea and Cercospora nicotianae and the bacterium Erwinia carotovora, but only marginally more to the bacterium Ralstonia solanacearum. In contrast, the biotrophic fungus Oidium neolycopersici, the oomycete Peronospora tabacina, and Tobacco mosaic virus caused similar or less severe symptoms on Tetr plants than on nontransformed plants. Total peroxidase activity of Tetr plants was lower than that of nontransformed plants, suggesting a role for peroxidases in resistance against necrotrophic microorganisms. A comparable range of pathogens was examined on Arabidopsis and its ethylene-insensitive mutants etr1-1 and ein2-1. With the exception of one Fusarium isolate, ethylene insensitivity increased susceptibility of Arabidopsis plants to a similar spectrum of necrotizing pathogens as in tobacco. Thus, both ethylene-insensitive tobacco and Arabidopsis plants appear to be impaired in their resistance to necrotrophic pathogens.

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10 7 CFU per g of roots soon after sowing to less than 10 2 CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.

Transgenic ethylene-insensitive tobacco (Tetr) plants spontaneously develop symptoms of wilting and stem necrosis when grown in nonautoclaved soil. Fusarium oxysporum, F. solani, Thielaviopsis basicola, Rhizopus stolonifer, and two Pythium spp. were isolated from these diseased Tetr plants and demonstrated to be causal agents of the disease symptoms. Pathogenicity of the two Pythium isolates and four additional Pythium spp. was tested on ethylene-insensitive tobacco and Arabidopsis seedlings. In both plant species, ethylene insensitivity enhanced susceptibility to the Pythium spp., as evidenced by both a higher disease index and a higher percentage of diseased plants. Based on the use of a DNA probe specific for Pythium spp., Tetr plants exhibited more pathogen growth in stem and leaf tissue than similarly diseased control plants. These results demonstrate that ethylene signaling is required for resistance to different root pathogens and contributes to limiting growth and systemic spread of the pathogen.

The First International Symposium on Induced Resistance to Plant Diseases, organized by Eris Tjamos, brought together over 150 participants to discuss the complexities, questions and future direction of research on the mechanisms by which plants can become better able to defend themselves against pathogen attack. Although the term immunization has been used to denote treatments that enhance the defensive capacity of plants, the correspondence to vaccination in vertebrates is far-fetched: the induced state is by no means specific, but rather constitutes a more general increase in plant resistance to various types of pathogens. Moreover, it seldom prevents disease from occurring but generally reduces its extent or severity. These characteristics make induced resistance a powerful mechanism to exploit for enhancing the overall resistance in crop plants. Indeed, the first commercial chemical triggering induced resistance in plants, acibenzolar-Smethyl (BTH) was recently introduced on the market by Novartis under the tradenames Actigard (USA) and BION (Europe).

Selected strains of rhizosphere bacteria reduce disease by activating a resistance mechanism in the plant named rhizobacteria-mediated induced systemic resistance (ISR). Rhizobacteria-mediated ISR resembles pathogeninduced systemic acquired resistance (SAR) in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of plant pathogens. Some rhizobacteria trigger the salicylic acid (SA)dependent SAR pathway by producing SA at the root surface. In other cases, rhizobacteria trigger a different signalling pathway that does not require SA. The existence of a SA-independent ISR pathway has been demonstrated in Arabidopsis thaliana. In contrast to pathogen-induced SAR, ISR induced by Pseudomonas fluorescensWCS417r is independent of SA accumulation and pathogenesis-related (PR) gene activation but, instead, requires responsiveness to the plant hormones jasmonic acid (JA) and ethylene. Mutant analyses showed that ISR follows a novel signalling pathway in which components from the JA and ethylene response are successively engaged to trigger a defensive state that, like SAR, is controlled by the regulatory factor NPR1. Interestingly, simultaneous activation of both the JA/ethylene-dependent ISR pathway and the SA-dependent SAR pathway results in an enhanced level of protection. Thus combining both types of induced resistance provides an attractive tool for the improvement of disease control. This review focuses on the current status of our research on triggering, signalling, and expression of rhizobacteria-mediated ISR in Arabidopsis.

Selected strains of nonpathogenic rhizobacteria from the genus Pseudomonas are capable of eliciting broad-spectrum induced systemic resistance (ISR) in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). In Arabidopsis, the ISR pathway functions independently of salicylic acid (SA) but requires responsiveness to jasmonate and ethylene. Here, we demonstrate that known defense-related genes, i.e. the SA-responsive genes PR-1, PR-2, and PR-5, the ethylene-inducible gene Hel, the ethylene- and jasmonate-responsive genes ChiB and Pdf1.2, and the jasmonate-inducible genes Atvsp, Lox1, Lox2, Pal1, and Pin2, are neither induced locally in the roots nor systemically in the leaves upon induction of ISR by Pseudomonas fluorescens WCS417r. In contrast, plants infected with the virulent leaf pathogen Pseudomonas syringae pv. tomato (Pst) or expressing SAR induced by preinfecting lower leaves with the avirulent pathogen Pst(avrRpt2) exhibit elevated expression levels of most of the defense-related genes studied. Upon challenge inoculation with Pst, PR gene transcripts accumulated to a higher level in SAR-expressing plants than in control-treated and ISR-expressing plants, indicating that SAR involves potentiation of SA-responsive PR gene expression. In contrast, pathogen challenge of ISR-expressing plants led to an enhanced level of Atvsp transcript accumulation. The other jasmonate-responsive defense-related genes studied were not potentiated during ISR, indicating that ISR is associated with the potentiation of specific jasmonate-responsive genes.

Fusarium wilt diseases, caused by the fungus Fusarium oxysporum, lead to significant yield losses of crops. One strategy to control fusarium wilt is the use of antagonistic, root-colonizing Pseudomonas spp. It has been demonstrated that different strains of these bacteria suppress disease by different mechanisms. Therefore, application of a mixture of these biocontrol strains, and thus of several suppressive mechanisms, may represent a viable control strategy. A prerequisite for biocontrol by combinations of biocontrol agents can be the compatibility of the co-inoculated micro-organisms. Hence, compatibility between several Pseudomonasspp. strains, that have the ability to suppress fusarium wilt of radish, was tested in vitro on KB agar plates. Growth of P. fluorescens strain RS111 was strongly inhibited by Pseudomonas spp. strains RE8, RS13, RS56 and RS158, whereas a mutant of strain RS111 (RS111-a) was insensitive to inhibition by these strains. Strains RS111 and RS111-a only slightly inhibited some other strains. Suppression of fusarium wilt of radish in a potting soil bioassay by the incompatible combination of RE8 and RS111 was comparable to the effects of the single strains. However, disease suppression by the compatible combination of RE8 and RS111-a was significantly better as compared to the single strains. In contrast, the incompatible combination of RS56 with RS111 resulted in enhanced disease suppression as compared to the single strains. Increased disease suppression by combinations of RS13 or RS158 with RS111 or RS111-a was not observed. This indicates that specific interactions between biocontrol strains influence disease suppression by combinations of these strains.

▪ Abstract Nonpathogenic rhizobacteria can induce a systemic resistance in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Rhizobacteria-mediated induced systemic resistance (ISR) has been demonstrated against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato under conditions in which the inducing bacteria and the challenging pathogen remained spatially separated. Bacterial strains differ in their ability to induce resistance in different plant species, and plants show variation in the expression of ISR upon induction by specific bacterial strains. Bacterial determinants of ISR include lipopolysaccharides, siderophores, and salicylic acid (SA). Whereas some of the rhizobacteria induce resistance through the SA-dependent SAR pathway, others do not and require jasmonic acid and ethylene perception by the plant for ISR to develop. No consistent host plant alterations are associated with the induced state, but upon challenge inoculation, resistance responses are accelerated and enhanced. ISR is effective under field conditions and offers a natural mechanism for biological control of plant disease.

To study the significance of stress-inducible ethylene in the hypersensitive reaction of tobacco (Nicotiana tabacum) to tobacco mosaic virus (TMV), six types of transgenic plants were generated containing sense or antisense constructs corresponding to the ethylene biosynthesis enzymes ACC-synthase (ACS) and/or ACC-oxidase under the control of the CaMV 35S promoter. In most primary transformants, expression of antisense RNA or incomplete sense RNA resulted in a reduction of inducible ethylene production, while plants overexpressing full-length sense ACS mRNA had higher ethylene levels than control plants. Although efficient overexpression or gene silencing was evident at the transcript level, there was no correlation with the amounts of ethylene produced, supporting the notion that ACS is post-transcriptionally regulated. Transgenic tobacco plants with altered ethylene levels were not impaired in their ability to respond with necrotic local lesions to infection with TMV. However, in the ACS sense and antisense transformants, alterations in ethylene levels did affect stem length and leaf chlorophyll levels in accordance with established ethylene responses.

Summary The primary leaves of 7- and 15-day-old oat plants were labelled with 35 S-methionine and the in vivo degradation of the synthesized proteins was followed by two-dimensional Polyacrylamide gel electrophoresis over a period of 2 weeks. During both leaf development and senescence protein breakdown comprised a rather quick turnover of a large number of proteins and a slow decline of a smaller number of persisting spots, many of which appeared to be chloroplast-associated. This pattern of breakdown was compared with that in vitro in soluble-protein extracts at pHs 5.5 and 7.5, the optimal pH values of the acidic and neutral proteases, respectively. Although 75% of the proteins were degraded similarly in vivo and in vitro , substantial differences in the disappearance of the other proteins and their relative rates of degradation occurred under the different conditions. Notably, ribulosebisphosphate carboxylase (Rubisco) was degraded very slowly in vivo , but rather quickly and yielding different degradation products in vitro at pH 5.5. In vivo protein degradation more closely resembled in vitro breakdown at pH 7.5. These results suggest that the vacuole has only a limited role in endogenous protein turnover and that several mechanisms are simultaneously active in protein degradation in oat leaves.

The nature of induced resistance Resistance, according to Agrios (1988) is the ability of an organism to exclude or overcome, completely or in some degree, the effect of a pathogen or other damaging factor. Disease resistance in plants is manifested by limited symptoms, reflecting the inability of the pathogen to grow or multiply and spread, and often takes the form of a hypersensitive reaction (HR), in which the pathogen remains confined to necrotic lesions near the site of infection. Induced resistance is the phenomenonthat a plant, once appropriately stimulated, exhibits an enhanced resistance upon 'challenge' inoculation with a pathogen. Although induced resistance has been attracting attention recently (Ryals et al., 1994; Hammerschmidt and Kuc, 1995), the first systematic enquiry into induced resistance was made by Ross (1961a,b). He observed that the inducible resistance response to tobacco mosaic virus (TMV) in N gene-containing, hypersensitively reacting tobacco was not confined to the immediate vicinity of the resulting local necrotic lesions, but extended to other plant parts. A ring of tissue around the developing lesions became fully refractory to subsequent infection (localized acquired resistance; Ross, 1961a), whereas challenge inoculation of distant tissues resulted in much smaller, and occasionally fewer, lesions (systemic acquired resistance (SAR); Ross, 1961b) than in non-induced plants. Even leaves thatweremere initials at the time of the primary inoculation became induced, suggesting that as a result of the initial infection, a signal was generated, transported and propagated, that primed the plant to respond more effectively to subsequent infection (Bozarth and Ross, 1964). Treatments that influenced lesion size after primary infection had similar effects on lesions developing upon challenge inoculation (Ross, 1966), leading to the conclusion that the mechanisms responsible for resistance expression were the same under both conditions. Only upon challenge inoculation, defense mechanisms appeared to be expressed earlier and to a greater extent (De Laat and Van Loon, 1983; Dean and Kuc, 1987).

The hypersensitive response of plants to infection by pathogens is associated with the induction of the expression of genes encoding pathogenesis-related (PR) proteins and the development of a systemic acquired resistance of the plant to viruses, fungi and bacteria. The PR genes induced in Samsun NN tobacco mosaic virus (TMV) have been classified into five groups, each encoding acidic extracellular and basic vacuolar proteins. In addition to induction by TMV, many PR genes are induced by treating the plant with salicylic acid (SA). Genes encoding acidic PRs are systemically induced upon TMV infection whereas genes encoding basic PRs are not. In contrast to the genes encoding acidic PRs, the genes encoding basic PRs are induced by ethylene and wounding. Cis-acting regulatory elements involved in the co-ordinate induction of PR genes by TMV and SA have been mapped in most detail in the PR-1 a promoter. This promoter contains a number of elements that bind a GT-1 like transcription factor with different affinities. To study the role of ethylene in the induction of PR gene expression, plants have been transformed with sense and antisense constructs of tobacco cDNAs encoding ACC-synthase and/or ACC-oxidase.

Conditions for UV-induced cross-linking of abscisic acid (ABA) through its enone chromophore to binding proteins were evaluated. The effects of a UV-light band between 260 and 530 nm on both unconjugated and protein-conjugated ABA, as well as on anti-ABA antibodies as models of ABA-binding proteins were determined. UV irradiation caused both isomerization and photolysis of ABA, but increasing the lower irradiation boundary to 345 nm strongly reduced photolysis and largely prevented isomerization. When conjugated to alkaline phosphatase (AP), ABA remained stable when using either a 320 or a 345 nm filter. At these wavelengths both binding of ABA to antibodies as well as AP enzymatic activity were maintained. UV-induced cross-linking of monoclonal anti-ABA antibodies to immobilized ABA was analysed by immunoassays. Optimal cross-linking was achieved after a 5 min irradiation period at 0°, using a long pass, cut-on filter to quench wavelengths below 290 nm. This cross-linking faithfully reflected cognate binding activity.

Pathogenesis-related proteins (abbreviated PRs) are defined as plant proteins that are induced in pathological or related situations. We propose a unifying nomenclature for PRs based on their grouping into families sharing amino acid sequences, serological relationship, and/or enzymatic or biological activity. The nomenclature classifies novel proteins identified by electrophoresis or chromatography along with those established by other workers. The previously proposed system of the five well-established families from tobacco is extended to accommodate a further six families. Families are indicated by arabic numerals and individual members are named by lower case letters in the order in which they are described. Additional rules are proposed to deal with forms containing more than a single polypeptide and as yet unclassified PRs. PR genes whose sequences are conserved but whose designations are not based on function are to be designated Ypr in accordance with the recommendations of the Commission on Plant Gene Nomenclature.

The rate of senescence and the two-dimensional pattern of soluble proteins from detached oat leaves senescing in either darkness or light were analyzed, and compared to those of leaves in which senescence was delayed by application of the cytokinin benzyladenine or enhanced through the action of abscisic acid.

Tobacco mosaic virus strains W U1, flavum, V4 and masked, which induce symptoms in tobacco ranging from a severe mosaic to virtually no reaction, were used to study relationships between accumulation of viral 126 kDa protein, X-body formation and symptom expression. The RNAs of all strains encode proteins of similar sizes to those of the common strain W U1. Antisera against the coat and 126 kDa proteins of W U1 recognized corresponding proteins of the other three strains. All virus strains accumulated to substantial levels in systemically infected leaves, as evidenced by the presence of coat protein in virus crystals. The 126 kDa protein was localized in large X-bodies associated with nuclei in W U1- and flavum-infected tissue, but in tiny X-bodies, free in the cytoplasm, in masked-infected plants. Neither X-bodies, nor 126 kDa proteins were observed in V4-infected tissue. Since in vitro synthesized 126 kDa protein from V4 was not more sensitive to proteolysis than that from W U1 or flavum, accumulation of the protein in X-bodies in the latter seems to result from excess synthesis. The possibility that the X-bodies are involved in the expression of severe symptoms is discussed.

In early botany, the study of morphogenesis in higher plants started at the level of the whole plant and its organs, describing their life cycle and the formation of the vegetative and generative organs during plant development. This stage of morphology was followed by that of physiology, in which experimental interference with developmental processes aimed at the analysis of causal relationships in plant morphogenesis. The past century offers beautiful examples of sophisticated experimental analysis of such developmental processes as, e.g., apical dominance and flower induction. It became apparent that plant metabolism, growth and development are genetically determined, and that the expression of the genetic information is dependent upon and modulated by internal and environmental factors such as, e.g., hormones and light. Neither the nature of the genes and the extent of their expression in particular developmental processes, nor the mechanism of action of the endogenous growth-regulating substances mediating internal and external signals were understood, however, although it seemed reasonable to assume that hormones influence developmental processes by bringing about changes in gene expression. Application of biochemical knowledge and methodology allowed for extension of experimentation at the cellular and subcellular levels, leading in several cases to the unravelling of hormonal or light-induced effects on enzyme activities in metabolic pathways. However, the significance of research at the protein level for the explanation of morphogenetic effects at the organ or whole-plant level has been severely limited by the correlative nature of the argumentation. The evidence is largely derived from simultaneously occurring variations at both levels, but apart from such correlations a firm causal argument cannot be obtained from combining physiological and biochemical data alone. Moreover, cell division, extension and differentiation, as well as the formation of organs and the spatial and temporal integration of various meristematic activities all depend on the interplay between various hormonal signals. Different combinations of hormones may act independently, synergistically, or antagonistically, and can influence each other’s metabolism, recognition by cellular receptors, or signal-transduction pathways. These complex interactions stem in part from the stochastic nature of hormonal regulation in plants itself (Trewavas 1991) and give rise to the plasticity of plant development, in which the pattern of growth and development is open to modulation by internal and external constraints and growth of new organs is invariably linked to loss of function of old organs (Woolhouse 1978).

Publisher Summary This chapter provides an overview of the processes and mechanisms. It has become clear that plants have evolved sophisticated mechanisms to deal with environmental challenges. To ward off attack by different types of organisms, they possess a surveillance system that recognizes common microbial components, such as bacterial lipopolysaccharides and flagellin, or fungal constituents comprising ergosterol and chitin. Plants flourish in almost all natural environments, even though they are surrounded by potentially harmful organisms. Because of their sessile nature, they have to cope effectively with these threats. During evolution they have developed effective mechanisms to counteract microbial invasion and animal attack. Apart from morphological adaptations, they rely on inducible defenses that are activated in response to infection or attack and limit proliferation of, and tissue colonization by the attacker.

Infection of plants with pathogens like viruses or fungi often leads to a response of the host ranging from very mild to very severe. A very mild response usually does not result in considerable changes in the patterns of gene expression of the plant. However, a hypersensitive reaction, e.g., in the case of infection of Samsun NN tobacco with tobacco mosaic virus, is accompanied by a massive increase in the amounts of a large number of different proteins. Among these induced proteins are members of a group of so‐called pathogenesis‐related (PR) proteins. Frequently, the hypersensitively reacting host becomes resistant to subsequent pathogenic attack. The correlation of this phenomenon of acquired resistance and the induced protein synthesis suggests a direct involvement of these proteins in the observed replication deficiency of the pathogen. This has initiated studies by several research groups toward a better understanding of the role of these PR‐proteins in the hypersensitively reacting plant....

Biological control of soilborne plant pathogens in the field has given variable results. By combining specific strains of microorganisms, multiple traits antagonizing the pathogen can be combined and this may result in a higher level of protection. Pseudomonas putida WCS358 suppresses Fusarium wilt of radish by effectively competing for iron through the production of its pseudobactin siderophore. However, in some bioassays pseudobactin-negative mutants of WCS358 also suppressed disease to the same extent as WCS358, suggesting that an, as yet unknown, additional mechanism may be operative in this strain. P. putida strain RE8 induced systemic resistance against fusarium wilt. When WCS358 and RE8 were mixed through soil together, disease suppression was significantly enhanced to approximately 50% as compared to the 30% reduction for the single strain treatments. Moreover, when one strain failed to suppress disease in the single application, the combination still resulted in disease control. The enhanced disease suppression by the combination of P. putida strains WCS358 and RE8 is most likely the result of the combination of their different disease-suppressive mechanisms. These results demonstrate that combining biocontrol strains can lead to more effective, or at least, more reliable biocontrol of fusarium wilt of radish.

Fluorescent Pseudomonas spp. have been studied for decades for their plant growth-promoting effects through effective suppression of soilborne plant diseases. The modes of action that play a role in disease suppression by these bacteria include siderophore-mediated competition for iron, antibiosis, production of lytic enzymes, and induced systemic resistance (ISR). The involvement of ISR is typically studied in systems in which the Pseudomonas bacteria and the pathogen are inoculated and remain spatially separated on the plant, e.g., the bacteria on the root and the pathogen on the leaf, or by use of split root systems. Since no direct interactions are possible between the two populations, suppression of disease development has to be plant-mediated. In this review, bacterial traits involved in Pseudomonas-mediated ISR will be discussed.

To protect themselves from disease, plants have evolved sophisticated defense mechanisms in which the signal molecules salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) often play crucial role. Elucidation of signaling pathways controlling disease resistance is a major objective in research on plant-pathogen interactions. The capacity of a plant to develop a broad spectrum, systemic acquired resistance (SAR) after primary infection with a necrotizing pathogen is well known and its signal transduction pathway extensively studied. Plants of which the roots have been colonized by specific strains of nonpathogenic fluorescent Pseudomonas spp. develop a phenotypically similar form of protection that is called rhizobacteria-mediated induced systemic resistance( ISR). In contrast to pathogen induced SAR, which is regulated by SA, rhizobacteria mediated ISR is controlled by a signaling pathway in which ET and JA play key roles.In the past decade, the model plant species Arabidopsis thaliana was explored to study the molecular basis of rhizobacteria mediated ISR. Here we review the current knowledge of the signal transduction steps involved in the ISR pathway that leads from recognition of the rhizobacteria in the roots to systemic expression of broad-spectrum disease resistance in above-ground foliar tissues.

Plant growth-promoting rhizobacteria can suppress diseases through antagonism between the bacteria and soil-borne pathogens, as well as by inducing a systemic resistance in the plant against both root and foliar pathogens. The generally non-specific character of induced resistance constitutes an increase in the level of basal resistance to several pathogens simultaneously, which is of benefit under natural conditions where multiple pathogens may be present. Specific Pseudomonas strains induce systemic resistance in e.g. carnation, cucumber, radish, tobacco and Arabidopsis, as evidenced by an enhanced defensive capacity upon challenge inoculation. Although some bacterial strains are equally effective in inducing resistance in different plant species, others show specificity, indicating specific recognition between bacteria and plants at the root surface. In carnation, radish and Arabidopsis, the O-antigenic side chain of the bacterial outer membrane lipopolysaccharide acts as an inducing determinant, but other bacterial traits are also involved. Pseudobactin siderophores have been implicated in the induction of resistance in tobacco and Arabidopsis, and another siderophore, pseudomonine, may explain induction of resistance associated with salicylic acid (SA) in radish. Although SA induces phenotypically similar systemic acquired resistance (SAR), it is not necessary for the systemic resistance induced by most rhizobacterial strains. Instead, rhizobacteria-mediated induced systemic resistance (ISR) is dependent on jasmonic acid (JA) and ethylene signaling in the plant. Upon challenge inoculation of induced Arabidopsis plants with a pathogen, leaves expressing SAR exhibit a primed expression of SA-, but not JA/ethylene-responsive defense-related genes, whereas leaves expressing ISR are primed to express JA/ethylene-, but not SA-responsive genes. Combination of ISR and SAR can increase protection against pathogens that are resisted through both pathways, as well as extend protection to a broader spectrum of pathogens than ISR or SAR alone.

Bacteria are by far the most abundant organisms in soil and they play a key role in nutrient cycling and soil fertility. The rhizosphere — the zone of 1–2 mm around plant roots — is rich in nutrients and provides niches different from those in bulk soil for bacteria to thrive. Microbial diversity in the soil and in the rhizosphere is huge. Multiple interactions occur between the bacteria and between bacteria and other microorganisms, involving competition, antibiosis, parasitism and predation. Various interactions also occur between bacteria and plant roots that can be beneficial, neutral or harmful to the plant. Deleterious effects comprise phytotoxic and pathogenic activities of the bacteria. Conversely, plants profit from bacterially induced growth promotion and protection against pathogens. Growth promotion can be the result of bacterial activities that increase the availability of water and mineral nutrients, as well as of symbiotic relationships such as the formation of root nodules in leguminous plants, in which atmospheric nitrogen is made available in reduced form. Nodulation of roots involves an intricate interplay of molecular signals between the bacterium and its host, and illustrates how such plant-rhizobacte-ria interactions proceed in an exquisitely controlled manner. In similar ways, suppression of disease-provoking microorganisms can occur through microbial antagonism in the rhizosphere as well as by specific interactions between the protective bacterium and its host.

Visker, M. H. P. W., Keizer, L. C. P., Budding, D. J., Van Loon, L. C., Colon, L. T., and Struik, P. C. 2003. Leaf position prevails over plant age and leaf age in reflecting resistance to late blight in potato. Phytopathology 93:666-674. The effects of plant age, leaf age, and leaf position on race-nonspecific resistance against Phytophthora infestans were investigated in a series of field and controlled environment experiments with five different potato (Solanum tuberosum) cultivars. Leaf position proved to be the most significant factor; apical leaves were far more resistant to late blight than basal leaves. Plant age and leaf age had only minor effects; therefore, the resistance of a specific leaf remained about the same during its entire lifetime. The gradual increase in late blight resistance from basal leaves to apical leaves appeared to be a general effect, irrespective of cultivar, growing conditions, or resistance test. Therefore, it is important to consider leaf position in tests for late blight resistance, because contrasts in resistance may be ascribed erroneously to differences between genotypes or treatments, whereas they are actually caused by differences in leaf position.

The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC , coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens , ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p -nitrophenyl-β- d - N , N ′, N "-triacetylchitotriose, but not on p -nitrophenyl-β- d - N -acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa , since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

12 pages, Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb EcoRI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida. Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs (pmsC and pmsB) showed homologies with chorismate-utilizing enzymes; a third ORF (pmsE) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene (pmsA). A putative promoter region and two predicted iron boxes were localized upstream of pmsC. We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli, deletions affecting the first ORF (pmsC) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production., Chemical Institute, University of Copenhagen, Copenhagen,Denmark and Hubrecht Laboratorium, Utrecht, The Netherland

Induced resistance is the phenomenon in which a plant, once appropriately stimulated, exhibits an enhanced resistance upon “challenge” inoculation with a pathogen. Induced resistance can be localized as well as systemic, and can be induced by limited pathogen infection, avirulent pathogens, certain non-pathogenic bacteria, and certain chemicals. Systemic induced resistance has been particularly well studied in tobacco, cucumber and Arabidopsis. Under the influence of the inducing stimulus, a mobile signal is generated and transported to other parts of the plant where it enhances the mechanisms normally functioning to limit infection, growth, multiplication and spread of fungi, bacteria and viruses. In the systemic acquired resistance (SAR) induced by pathogens causing hypersensitive necrosis, resistance mechanisms to challenge inoculation become operative earlier or operate at a higher level than in non-induced plants. The expression of SAR is dependent on the accumulation of salicylic acid (SA) and associated with the induction of pathogenesis-related proteins (PRs) with anti-pathogen activities. In contrast, rhizobacterially-mediated induced systemic resistance (ISR) does not require SA, can occur without production of PRs, and is dependent on ethylene and jasmonic acid (JA) signalling. Mutants in the signal-transduction pathways of SAR and ISR have been identified in Arabidopsis which point to both pathways converging at the final step, while additional antimicrobial peptides are independently regulated. A different type of systemic induced resistance, involving production of proteinase inhibitors against herbivores, also requires JA. Synthesis of JA is initiated on perception of the Polypeptide systemin, which is released on wounding of tomato and potato and acts as the mobile signal. These various types of systemic induced resistance confer an enhanced defensive capacity on plants against a broad spectrum of attackers and offer great potential for exploitation in crop protection.

Download : Download full-size image Various rhizobacterial strains suppress soil-borne plant diseases by competition with the pathogen for nutrients, siderophore-mediated competition for iron, antibiosis, or production of lytic enzymes. Some strains can also enhance the defensive capacity of plants by mechanisms that are either dependent on the production of salicylic acid and associated with the accumulation of pathogenesis-related proteins in the plant (systemic acquired resistance), or that are not but require responsiveness of the plant to jasmonate and ethylene (induced systemic resistance). To exploit the mechanisms involved in disease suppression by rhizobacteria, the genes responsible are being cloned. Combinations of rhizobacterial strains or genetic modification of Pseudomonas bacteria are used to improve biological control of economically important plant diseases. Risk assessment studies have been initiated with regard to the introduction of such genetically-modified microorganisms in the field.

In incompatible host-pathogen interactions, damage caused by the pathogen remains restricted as a result of the plant's defensive response. Most effective is the hypersensitive reaction, in which the cells around the infection site rapidly necrose. This response is associated with a coordinated and integrated set of metabolic alterations that are instrumental in impeding further pathogen ingress, as well as in enhancing the capacity of the host to limit subsequent infection by different types of pathogens [27, 77]. Altered ion fluxes across the plant cell membrane, generation of active oxygen species, changes in the phosphorylation state of regulatory proteins and transcriptional activation of plant defense systems culminate in cell death at the site of infection, local accumulation of phytoalexins and cell wall rigidification as a result of callose, lignin and suberin deposition [31, 89]. In addition, various novel proteins are induced which are collectively referred to as ``pathogenesis-related proteins '' (PRs). These PRs, defined as proteins coded for by the host plant but induced specifically in pathological or related situations [4, 81], do not only accumulate locally in the infected leaf, but are also induced systemically, associated with the development of systemic acquired resistance (SAR) against further infection by fungi, bacteria and viruses. Induction of PRs has been found in many plant species belonging to various families [78], suggestive of a general role for these proteins in adaptation to biotic stress conditions. SAR, likewise, is a generally occurring phenomenon, that engenders an enhancement of the defensive capacity of plants in response to necrotizing infections [70]. Since some of the tobacco PRs were identified as chitinases [45] and b-1,3-glucanases [38] with potential antifungal activity, it has often been suggested that the collective set of PRs may be effective in inhibiting pathogen growth, multiplication and/or spread, and be responsible for the state of SAR [42, 65]. Originally, five main classes of PRs (PR-1-5) were characterized by both biochemical and molecular-biological techniques in tobacco [9, 80]. Thereupon, in 1994 a unifying nomenclature for PRs was proposed based on their grouping into families sharing amino acid sequences, serological relationship, and/or enzymatic or biological activity. By then 11 families (PR-1--11) were recognized and classified for tobacco and tomato [81] (cf. Table 1). Criteria used for the inclusion of new families of PRs were that (i) protein(s) must be induced by a pathogen in tissues that do not normally express the protein(s), and (ii) induced expression must have been shown to occur in at least two different plant-pathogen combinations, or expression in a single plant-pathogen combination must have been confirmed independently in diaerent laboratories.

Ethylene is involved in many physiological and developmental processes in plants, as well as in the responses of plants to abiotic and biotic stresses [1, 2]. In hypersensitively reacting tobacco, tobacco mosaic virus (TMV) infection results in increased ethylene synthesis [3]. Associated with TMV infection is the production of acidic and basic pathogenesis-related (PR)-proteins several of which have antifungal activity [4, 5, 6]. Associated with the production of PR-proteins, the plant becomes more resistant to subsequent pathogen attack, a phenomenon known as systemic acquired resistance (SAR) [7, 8]. Under the influence of the primary infection, a signal moves through the plant and induces the resistant state.

In contrast to wild-type seeds of Arabidopsis thaliana and to seeds deficient in (aba) or insensitive to (abi3) abscisic acid (ABA), maturing seeds of recombinant (aba,abi3) plants fail to desiccate, remain green, and lose viability upon drying. These double-mutant seeds acquire only low levels of the major storage proteins and are deficient in several low mol wt polypeptides, both soluble and bound, and some of which are heat stable. A major heat-stable glycoprotein of more than 100 kilodaltons behaves similarly; during seed development, it shows a decrease in size associated with the abi3 mutation. In seeds of the double mutant from 14 to 20 days after pollination, the low amounts of various maturation-specific proteins disappear and many higher mol wt proteins similar to those occurring during germination are induced, but no visible germination is apparent. It appears that in the aba,abi3 double mutant seed development is not completed and the program for seed germination is initiated prematurely in the absence of substances protective against dehydration. Seeds may be made desiccation tolerant by watering the plants with the ABA analog LAB 173711 or by imbibition of isolated immature seeds, 11 to 15 days after pollination, with ABA and sucrose. Whereas sucrose stimulates germination and may protect dehydration-sensitive structures from desiccation damage, ABA inhibits precocious germination and is required to complete the program for seed maturation and the associated development of desiccation tolerance.

Summary Of the three molecular forms of petunia (Petunia hybrida) peroxidase a, the form PRXa 1.1 (37.5 kDa) was retarded on columns of concanavalin A-Sepharose, whereas forms PRXa 1.2 and 1.3 (37.0 and 36.5 kDa, respectively) were not. Chemical deglycosylation of the two fractions yielded only poorly resolved products of 33–35 kDa. Enzymatic deglycosylation with c-mannosidase converted PRXa 1.1 and 1.2 to immunoreactive products migrating in polyacrylamide gels at the position of PRXa 1.3. The presence of xylose, arabinose, fucose, mannose, glucose, and galactose was indicated by gas- chromatographic analysis. Lectin affinity blotting using lectins conjugated to horseradish peroxidase suggested that PRXa contains small complex-type N- glycosyl carbohydrate chains. These results indicate that the heterogeneity of PRXa is caused by differences in the terminal a-linked mannose residues. Some ce-mannosidase activity was present in the apoplast of leaves, which is consistent with the suggestion that it may be responsible for the conversion of PRXa 1.1 into PRXa 1.2 and 1.3 during leaf development.

Summary The distribution of soluble peroxidase activity in tissues from petals, leaves and stems of petunia ( Petunia hybrida ) was investigated. In petals, the upper epidermis contained nearly 20 % of the soluble peroxidase activity, but no peroxidase activity was recovered from protoplasts isolated from this tissue. In contrast, protoplasts from the remaining tissue contained about 45 % of the total peroxidase activity in the form of the isoenzyme PRXb. The extracellular peroxidase activity present in the epidermis was mainly PRXa. In leaves, the lower epidermis contained 14-40 % of the soluble peroxidase activity, depending on their stage of development. In fully expanded leaves about half of the activity in the lower epidermis was located extracellularly. In stems, nearly 80 % of the soluble peroxidase activity was present in the epidermis, and about 30 % of this activity was localized extracellularly. Since the extracellular extracts contained merely PRXa, these results suggested that PRXa is localized mainly in the epidermis of the aerial organs of petunia. When leaves and stems were cut transversely and pressed on membrane filters, the resulting dip-blots were employed for in situ localization of peroxidase by activity staining and of PRXa by immunodetection. Both peroxidase activity and immunoreactive PRXa were present mainly in the epidermis of stems and leaves, particularly at the site of the midrib and veins. With respect to this distribution, the function of PRXa is discussed.

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and F. occidentalis, more than 50% also were induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plant’s defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.