Nontarget and ecological effects of transgenically altered disease resistance in crops -possible effects on the mycorrhizal symbiosis

The ability to increase crop disease resistance by using transgenic (TG) means has recently been demonstrated for several crops. The current TG procedures alter the temporal expression of transgene pathogenesis-related (PR) proteins, so that the usually inducible PR proteins are expressed constitutively in the foreign host. The constitutive expression of the transgene PR protein chitinase is believed to increase the host's nonspecific basic resistance to pathogens. A potential nontarget effect of constitutively expressing chitinase may be a decrease in the activity of beneficial microbes, especially vesicular-arbuscular mycorrhizal fungi. The decrease in activity of mycorrhizal fungi is related to reduced susceptibility of TG plant roots to colonization by these fungi, which is in turn associated with lysis of fungal cell walls by the constitutively expressed chitinase. An argument is presented that use of TG means to alter the temporal expression of PR proteins ignores a legacy of past evolutionary trade-offs in vascular plants. A major nontarget effect of expressing transgene chitinase is a reduction in the susceptibility of roots to colonization by mycorrhizal fungi. This reduction in mycorrhizal susceptibility occurs without alteration of the mycorrhizal dependence of the host on symbiont-supplied nutrients. Data are presented in support of this contention that demonstrate a strong negative association between host pathogen resistance and mycorrhizal colonization. An ecological consequence of reducing mycorrhizal colonization is a decrease in the soil's mycorrhizal propagule reserve that diminishes the next crop's production, especially under low-input cropping practices. A further consequence that has both ecological and evolutionary outcomes is the escape of the transgene for improved pathogen resistance into wild populations. By increasing a crop's disease resistance by TG means, we may inadvertently be creating a ‘super weed’ when the TG plant or the transgene escapes into wild relatives through hybridization. Hybridization of wild relatives with TG plants would be especially relevant for crops, such as sugar beet, rapeseed, and many modern cereal cultivars that have close relatives in the wild but have a relatively low requirement for symbiont supplied nutrients or are nondependent.     

Regulation of defense and cryoprotective proteins by high levels of CO(2) in Annona fruit stored at chilling temperature

This study focuses on how the length of exposure to chilling temperature and atmosphere storage conditions regulate the hydrolytic activity and expression of chitinase (PR-Q) and 1,3-beta-glucanase (PR-2) isoenzymes in cherimoyas (Annona cherimola Mill.). Storage at 6 degrees C modified the expression of constitutive isoenzymes and induced the appearance of novel acidic chitinases, AChi26 and AChi24, at the onset of the storage period, and of a basic chitinase, BChi33, after prolonged storage. The induction of this basic isoenzyme was concomitant with the accumulation of basic constitutive 1,3-beta-glucanases. These low-temperature-induced chitinases modified the growth inhibition in vitro of Botrytis cinerea. Short-term high CO(2) treatment activated a coordinated response of acidic chitinases and 1,3-beta-glucanases after prolonged storage at chilling temperature. Moreover, the high in vitro cryoprotective activity of CO(2)-treated protein extracts was associated with the induction of two low molecular mass isoenzymes, AGlu19 and BChi14. Thus, exposure to high concentrations of CO(2) modified the response of fruit to low temperature, inducing the synthesis of cryoprotectant proteins such as specific pathogenesis-related isoenzymes that could be functionally associated with an increase in chilling tolerance in vivo.     

Interactions between engineered tomato plants expressing antifungal enzymes and nontarget fungi in the rhizosphere and phyllosphere

The introduction of genetically modified (GM) plants in agroecosystems raises concern about possible effects on nontarget species. The impact of a tomato line transformed for constitutive expression of tobacco beta-1,3-glucanase and chitinase on indigenous nonpathogenic fungi was investigated. In greenhouse experiments, no significant differences were found in the colonization by arbuscular mycorrhizal fungi. Diversity indices computed from over 20 500 colonies of culturable rhizosphere and phyllosphere saprotrophic microfungi, assigned to 165 species (plus > 80 sterile morphotypes), showed no significant differences between GM and wild-type plants. Differences were found by discriminant analysis in both the rhizosphere and the phyllosphere, but such effects were minor compared with those linked to different plant growth stages.     

Tobacco genes encoding acidic and basic isoforms of pathogenesis-related proteins display different expression patterns

The induction by cytokinin stress and ethylene of nine different tobacco mosaic virus-inducible mRNA classes (termed A-I) encoding pathogenesis-related (PR) proteins was studied. The induced mRNA levels were compared to basal levels in healthy tobacco plants grown in tissue culture and in a greenhouse. Cytokinin stress and ethylene were found to induce different subsets of the mRNAs, indicating that ethylene is not the primary inducing signal in cytokinin-stressed shoots. mRNAs F, H and G encoding the basic hydrolytic enzymes chitinase, -1,3-glucanase and a basic equivalent of PR-1, respectively, were found to be expressed at high levels in roots of healthy plants. mRNAs D, I and B encoding the acidic equivalents of the proteins proved to be present at low levels in healthy plants. These results indicate that genes encoding basic and acidic isoforms of pathogenesis-related proteins are differentially regulated.     

Benzothiadiazole as an Inducer of β-1,3-Glucanase and Chitinase Isozymes in Sugar Beet

The effect of benzothiadiazole (BTH) on protein synthesis was studied in sugar beet plants. Extracellular proteins induced by 0.025 % BTH were examined and their pattern was compared with that induced by sodium salicylate, chitosan, paraquat, AgNO₃, and by tobacco necrosis virus. BTH induced synthesis of at least 9 acidic and 6 basic proteins; three of them appeared as acidic chitinase isozymes, three as acidic β-1,3-glucanase isozymes, three as basic chitinase isozymes, and one as a basic β-1,3-glucanase isozyme. One of the basic chitinase isozymes was found also in control plants. The most of the newly formed proteins was also induced by the other inducers under study regardless of the necrotic or symptomless reaction of plants. The benzothiadiazole proved to be an efficient inducer of proteins in sugar beet. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.     

Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity

Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.     

Aphid infestation induces PR-proteins differently in barley susceptible or resistant to the birdcherry-oat aphid (Rhopalosiphum padi)

The effect of infestation by the birdcherry-oat aphid ( Rhopalosiphum padi L.), on induction of PR-proteins was investigated in barley ( Hordeum vulgare L.), using barley lines susceptible or resistant to R. padi. The PR-proteins PR-1a (unknown function), PR-5a (acidic thaumatin) and peroxidase (EC 1.11.1.7) were not affected, whereas one chitinase (EC 3.2.1.14) and 4 β -1,3-glucanases (EC 3.2.1.39) were induced by the aphid treatment. In the resistant breeding line CI 16145, but not in the susceptible cultivar Golf, accumulation of one basic chitinase and two acidic β -1,3-glucanases increased with time from 2 until 11 days after infestation, as determined by western blots, with antibodies raised against purified chitinase (PR-3a) and β -1,3-glucanase (PR-2a) from barley. By isoelectric focusing, two additional basic β -1,3-glucanases were detected, which increased after infestation in both the resistant and the susceptible barley. The basic chitinase was only detected at days 7 and 11 in the susceptible cultivar, but already at day 2 in the resistant line. The induction was localized to the infested leaf. The PR-proteins PR-3a and PR-2a were also induced by the fungal pathogen ( Blumeria [syn. Erysiphe ] graminis f. sp. hordei ), methyl salicylate and, to a lower extent, by wounding with tweezers and methyl jasmonate (MeJA). Needle wounding performed to mimic aphid stylet penetration did not induce chitinase or β -1,3-glucanase. It is concluded that the fungal pathogen and the aphid infestation induce both similar and different responses, and that the aphid induction is not due to wounding only. The different responses in resistant and susceptible lines indicate that the induced enzymes may play a role in the resistance against aphid infestation.     

AM真菌和胞囊线虫对大豆根内酶活性的影响*

将‘鲁豆4号’大豆接种丛枝菌根(AM)真菌聚生球囊霉Glomus fasiculatum和大豆胞囊线虫(SCN)Heterodera glycines 4号生理小种后, 定期测定大豆根系中AM真菌及线虫侵染速率、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、β-1,3葡聚糖酶及几丁质酶活性的动态变化。结果表明, 接种AM真菌大豆根系中4种酶活性高于对照水平; 先接种AM真菌后接种SCN处理根系中POD、PAL及几丁质酶的活性高于只接种SCN的处理,并且酶活性峰值出现的时间均早于或相当于后者。另外,PAL及几丁质酶活性出现高峰时期也正是AM真菌侵染率迅速升高及线虫侵染速率快速下降期。因此,AM真菌先激活了大豆的防御反应,然后使其对SCN的侵染产生快速反应,PAL及几丁质酶在AM真菌诱导的抗、耐线虫病害机制中起重要作用。值得注意的是,先接种AM真菌后接种SCN处理大豆根系中,β-1,3葡聚糖酶活性低于只接种AM真菌的处理。作者认为本试验条件下,该酶在大豆抗SCN病害中的作用表现不明显。     

Ozone-induced changes of mRNA levels of β-1,3-glucanase,chitinase and ‘pathogenesis-related’ protein 1b in tobacco plants

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 l/l, 5 h) markedly increased the mRNA level of basic -1,3-glucanase and to a lower degree that of basic chitinase. The increase of -1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the -1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of -1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of -1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of -1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and pathogenesis-related (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.     

Localized versus systemic effect of arbuscular mycorrhizal fungi on defence responses to Phytophthora infection in tomato plants

Development of biological control for plant diseases is accepted as a durable and environmentally friendly alternative for agrochemicals. Arbuscular mycorrhizal fungi (AMF), which form symbiotic associations with root systems of most agricultural, horticultural and hardwood crop species, have been suggested as widespread potential bioprotective agents. In the present study the ability of two AMF (Glomus mosseae and Glomus intraradices) to induce local or systemic resistance to Phytophthora parasitica in tomato roots have been compared using a split root experimental system. Glomus mosseae was effective in reducing disease symptoms produced by P. parasitica infection, and evidence points to a combination of local and systemic mechanisms being responsible for this bioprotector effect. The biochemical analysis of different plant defence-related enzymes showed a local induction of mycorrhiza-related new isoforms of the hydrolytic enzymes chitinase, chitosanase and beta-1,3-glucanase, as well as superoxide dismutase, an enzyme which is involved in cell protection against oxidative stress. Systemic alterations of the activity of some of the constitutive isoforms were also observed in non-mycorrhizal roots of mycorrhizal plants. Studies on the lytic activity against Phytophthora cell wall of root protein extracts also corroborated a systemic effect of mycorrhizal symbiosis on tomato resistance to Phytophthora.     

beta]-1,3-Glucanase Is Cryoprotective in Vitro and Is Accumulated in Leaves during Cold Acclimation

We have used isolated spinach (Spinacea oleracea L.) thylakoid membranes to investigate the possible cryoprotective properties of class I [beta]-1,3-glucanase (1,3-[beta]-D-glucan 3-glucanohydrolase; EC 3.2.1.39) and chitinase. Class I [beta]-1,3-glucanase that was purified from tobacco (Nicotiana tabacum L.) protected thylakoids against freeze-thaw injury in our in vitro assays, whereas class I chitinase from tobacco had no effect under the same conditions. The [beta]-1,3-glucanase acted by reducing the influx of solutes into the membrane vesicles during freezing and thereby reduced osmotic stress and vesicle rupture during thawing. Western blots probed with antibodies directed against tobacco class I [beta]-1,3-glucanase showed that in spinach and cabbage (Brassica oleracea L.) leaves an isoform of 41 kD was accumulated during frost hardening under natural conditions.     

Differential Regulation of beta-1,3-Glucanase Messenger RNAs in Response to Pathogen Infection

The acidic, extracellular, glucan endo-1,3-β-glucosidases (EC 3.2.1.39; β-1,3-glucanases), pathogenesis-related proteins-2, -N, and -O (i.e. PR-2, PR-N, and PR-O) were purified from Nicotiana tabacum (tobacco) and their partial amino acid sequences determined. Based on these data, complementary DNA (cDNA) clones encoding the proteins were isolated. Additional cDNAs were isolated that encoded proteins approximately 90% identical with PR-2, PR-N, and PR-O. Although the proteins encoded by these cDNAs have not been identified, their deduced amino acid sequences have slightly basic or neutral calculated isoelectric points, as well as carboxy-terminal extensions. These physical characteristics are shared by the vacuolar form of β-1,3-glucanase and other vacuolar localized analogs of PR proteins, suggesting that the unidentified proteins may be similarly localized. A preliminary evolutionary model that separates the β-1,3-glucanase gene family from tobacco into at least five distinct subfamilies is proposed. The expression of β-1,3-glucanase messenger RNAs (mRNAs) in response to infection by tobacco mosaic virus was examined. Messages for the acidic glucanases were induced similarly to the mRNAs for other PR proteins. However, the basic glucanase showed a different response, suggesting that different isoforms are differentially regulated by tobacco mosaic virus infection at the mRNA level.     

Silencing of acidic pathogenesis-related PR-1 genes increases extracellular beta-(1->3)-glucanase activity at the onset of tobacco defence reactions

The class 1 pathogenesis-related (PR) proteins are thought to be involved in plant defence responses, but their molecular functions are unknown. The function of PR-1 was investigated in tobacco by generating stable PR-1a-silenced lines in which other acidic PR-1 genes (PR-1b and PR-1c) were silenced. Plants lacking extracellular PR-1s were more susceptible than wild-type plants to the oomycete Phytophthora parasitica but displayed unaffected systemic acquired resistance and developmental resistance to this pathogen. Treatment with salicylic acid up-regulates the PR-1g gene, encoding a basic protein of the PR-1 family, in PR-1-deficient tobacco, indicating that PR-1 expression may repress that of PR-1g. This shows that acidic PR-1s are dispensable for expression of salicylic acid-dependent acquired resistances against P. parasitica and may reveal a functional overlap in tobacco defence or a functional redundancy in the PR-1 gene family. The data also show that there is a specific increase in apoplastic beta-(1-->3)-glucanase activity and a decrease in beta-(1-->3)-glucan deposition in PR-1-silenced lines following activation of defence reactions. Complementation of the silencing by apoplastic treatment with a recombinant PR-1a protein largely restores the wild-type beta-(1-->3)-glucanase activity and callose phenotype. Taken together with the immunolocalization of PR-1a to sites of beta-(1-->3)-glucan deposition in wild-type plants, these results are indicative of a function for PR-1a in regulation of enzymatic activity of extracellular beta-(1-->3)-glucanases.     

Interplant communication of tomato plants through underground common mycorrhizal networks

Plants can defend themselves to pathogen and herbivore attack by responding to chemical signals that are emitted by attacked plants. It is well established that such signals can be transferred through the air. In theory, plants can also communicate with each other through underground common mycorrhizal networks (CMNs) that interconnect roots of multiple plants. However, until now research focused on plant-to-plant carbon nutrient movement and there is no evidence that defense signals can be exchanged through such mycorrhizal hyphal networks. Here, we show that CMNs mediate plant-plant communication between healthy plants and pathogen-infected tomato plants (Lycopersicon esculentum Mill.). After establishment of CMNs with the arbuscular mycorrhizal fungus Glomus mosseae between tomato plants, inoculation of 'donor' plants with the pathogen Alternaria solani led to increases in disease resistance and activities of the putative defensive enzymes, peroxidase, polyphenol oxidase, chitinase, β-1,3-glucanase, phenylalanine ammonia-lyase and lipoxygenase in healthy neighbouring 'receiver' plants. The uninfected 'receiver' plants also activated six defence-related genes when CMNs connected 'donor' plants challenged with A. solani. This finding indicates that CMNs may function as a plant-plant underground communication conduit whereby disease resistance and induced defence signals can be transferred between the healthy and pathogen-infected neighbouring plants, suggesting that plants can 'eavesdrop' on defence signals from the pathogen-challenged neighbours through CMNs to activate defences before being attacked themselves.     

Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea

Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola results in systemic induction of genes encoding a plant defensin (PDF1.2), a basic chitinase (PR-3), and an acidic hevein-like protein (PR-4). Pathogen-induced induction of these three genes is almost completely abolished in the ethylene-insensitive Arabidopsis mutant ein2-1. This indicates that a functional ethylene signal transduction component (EIN2) is required in this response. The ein2-1 mutants were found to be markedly more susceptible than wild-type plants to infection by two different strains of the gray mold fungus Botrytis cinerea. In contrast, no increased fungal colonization of ein2-1 mutants was observed after challenge with avirulent strains of either Peronospora parasitica or A. brassicicola. Our data support the conclusion that ethylene-controlled responses play a role in resistance of Arabidopsis to some but not all types of pathogens.