Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1–1Δ mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1–1Δ mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1–1Δ mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.

Fusicoccin, 14-3-3 Proteins, and Defense Responses in Tomato Plants1

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.

A Benzothiadiazole Primes Parsley Cells for Augmented Elicitation of Defense Responses

Systemic acquired resistance is an important component of the disease-resistance arsenal of plants, and is associated with an enhanced potency for activating local defense responses upon pathogen attack. Here we demonstrate that pretreatment with benzothiadiazole (BTH), a synthetic activator of acquired resistance in plants, augmented the sensitivity for low-dose elicitation of coumarin phytoalexin secretion by cultured parsley (Petroselinum crispum L.) cells. Enhanced coumarin secretion was associated with potentiated activation of genes encoding Phe ammonia-lyase (PAL). The augmentation of PAL gene induction was proportional to the length of pretreatment with BTH, indicating time-dependent priming of the cells. In contrast to the PAL genes, those for anionic peroxidase were directly induced by BTH in the absence of elicitor, thus confirming a dual role for BTH in the activation of plant defenses. Strikingly, the ability of various chemicals to enhance plant disease resistance correlated with their capability to potentiate parsley PAL gene elicitation, emphasizing an important role for defense response potentiation in acquired plant disease resistance.

Defense Responses to Tetrapyrrole-Induced Oxidative Stress in Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or Coproporphyrinogen Oxidase Activity1

We analyzed the antioxidative defense responses of transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase. These plants are characterized by necrotic leaf lesions resulting from the accumulation of potentially photosensitizing tetrapyrroles. Compared with control plants, the transformants had increased levels of antioxidant mRNAs, particularly those encoding superoxide dismutase (SOD), catalase, and glutathione peroxidase. These elevated transcript levels correlated with increased activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD. Total catalase activity decreased in the older leaves of the transformants to levels lower than in the wild-type plants, reflecting an enhanced turnover of this photosensitive enzyme. Most of the enzymes of the Halliwell-Asada pathway displayed increased activities in transgenic plants. Despite the elevated enzyme activities, the limited capacity of the antioxidative system was apparent from decreased levels of ascorbate and glutathione, as well as from necrotic leaf lesions and growth retardation. Our data demonstrate the induction of the enzymatic detoxifying defense system in several compartments, suggesting a photosensitization of the entire cell. It is proposed that the tetrapyrroles that initially accumulate in the plastids leak out into other cellular compartments, thereby necessitating the local detoxification of reactive oxygen species.

Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses.

In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of H2O2, which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxoidase (APX), the other key enzyme for scavenging H2O2. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 microM ascorbic acid (AsA), substrate-dependent IC50 values of 78 microM and 95 microM were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated H2O2 levels or coupled perturbations of the cellular redox state.

Two inducers of plant defense responses, 2,6-dichloroisonicotinec acid and salicylic acid, inhibit catalase activity in tobacco.

2,6-Dichloroisonicotinic acid (INA) and salicylic acid (SA) are potent inducers of plant defense responses including the synthesis of pathogenesis-related (PR) proteins and the development of enhanced disease resistance. A soluble SA-binding protein has been purified from tobacco with an affinity and specificity of binding that suggest it is a SA receptor. Recently, this protein has been shown to be a catalase whose enzymatic activity is inhibited by SA binding. We have proposed that the resulting increase in intracellular levels of reactive oxygen species plays a role in the induction of defense responses such as PR protein gene expression. Here we report that INA, like SA, binds the SA-binding protein/catalase and inhibits its enzymatic activity. In fact, the dose-response curves for inhibition of catalase by these two compounds are similar. Furthermore, the ability of both INA analogues and SA derivatives to bind and inhibit tobacco catalase correlates with their biological activity to induce PR-1 gene expression and enhance resistance to tobacco mosaic virus. Comparison of the structures of INA, SA, and their analogues reveals several common features that appear to be important for biological activity. Thus, these results not only suggest that INA and SA share the same mechanism of action that involves binding and inhibition of catalase but also further indicate an important role for reactive oxygen species in the induction of certain plant defense responses. This is supported by the demonstration that INA-mediated PR-1 gene activation is suppressed by antioxidants.

Induction, modification, and transduction of the salicylic acid signal in plant defense responses.

Studies in our laboratory as well as others strongly suggest that salicylic acid (SA) plays an important signaling role in plant defense against pathogens. We have found that increases in endogenous SA levels correlates with both resistance of tobacco to infection with tobacco mosaic virus and induction of defense-related genes such as that encoding pathogenesis-related protein 1 (PR-1). Some of this newly synthesized SA was conjugated to glucose to form SA beta-glucoside. A cell wall-associated beta-glucosidase activity that releases SA from this glucoside has been identified, suggesting that SA beta-glucoside serves as an inactive storage form of SA. By purifying a soluble SA-binding protein and isolating its encoding cDNA from tobacco, we have been able to further characterize the mechanism of SA signaling. This protein is a catalase, and binding of SA and its biologically active analogues inhibited catalase's ability to convert H2O2 to O2 and H2O. The resulting elevated levels of cellular H2O2 appeared to induce PR-1 gene expression, perhaps by acting as a second messenger. Additionally, transgenic tobacco expressing an antisense copy of the catalase gene and exhibiting depressed levels of catalase also showed constitutive expression of PR-1 genes. To further dissect the SA signaling pathway, we have tested several abiotic inducers of PR gene expression and disease resistance for their ability to stimulate SA production. Levels of SA and its glucoside rose following application of all of the inducers except 2,6-dichloroisonicotinic acid. 2,6-Dichloroisonicotinic acid was found to bind catalase directly and inhibit its enzymatic activity. Thus, it appears that many compounds that induce PR gene expression and disease resistance in plants inactivate catalases directly or indirectly.

Induction of Defense-Related Responses in Cf9 Tomato Cells by the AVR9 Elicitor Peptide of Cladosporium fulvum Is Developmentally Regulated1

The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene. To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated. Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes. Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation. Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained. Transgenic F1 seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene. It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells. These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.

Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense

Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungus Alternaria alternata in the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.

Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana

The plant-signaling molecules salicylic acid (SA) and jasmonic acid (JA) play an important role in induced disease resistance pathways. Cross-talk between SA- and JA-dependent pathways can result in inhibition of JA-mediated defense responses. We investigated possible antagonistic interactions between the SA-dependent systemic acquired resistance (SAR) pathway, which is induced upon pathogen infection, and the JA-dependent induced systemic resistance (ISR) pathway, which is triggered by nonpathogenic Pseudomonas rhizobacteria. In Arabidopsis thaliana, SAR and ISR are effective against a broad spectrum of pathogens, including the foliar pathogen Pseudomonas syringae pv. tomato (Pst). Simultaneous activation of SAR and ISR resulted in an additive effect on the level of induced protection against Pst. In Arabidopsis genotypes that are blocked in either SAR or ISR, this additive effect was not evident. Moreover, induction of ISR did not affect the expression of the SAR marker gene PR-1 in plants expressing SAR. Together, these observations demonstrate that the SAR and the ISR pathway are compatible and that there is no significant cross-talk between these pathways. SAR and ISR both require the key regulatory protein NPR1. Plants expressing both types of induced resistance did not show elevated Npr1 transcript levels, indicating that the constitutive level of NPR1 is sufficient to facilitate simultaneous expression of SAR and ISR. These results suggest that the enhanced level of protection is established through parallel activation of complementary, NPR1-dependent defense responses that are both active against Pst. Therefore, combining SAR and ISR provides an attractive tool for the improvement of disease control.

Jasmonic acid carboxyl methyltransferase: A key enzyme for jasmonate-regulated plant responses

Methyl jasmonate is a plant volatile that acts as an important cellular regulator mediating diverse developmental processes and defense responses. We have cloned the novel gene JMT encoding an S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT) from Arabidopsis thaliana. Recombinant JMT protein expressed in Escherichia coli catalyzed the formation of methyl jasmonate from jasmonic acid with Km value of 38.5 μM. JMT RNA was not detected in young seedlings but was detected in rosettes, cauline leaves, and developing flowers. In addition, expression of the gene was induced both locally and systemically by wounding or methyl jasmonate treatment. This result suggests that JMT can perceive and respond to local and systemic signals generated by external stimuli, and that the signals may include methyl jasmonate itself. Transgenic Arabidopsis overexpressing JMT had a 3-fold elevated level of endogenous methyl jasmonate without altering jasmonic acid content. The transgenic plants exhibited constitutive expression of jasmonate-responsive genes, including VSP and PDF1.2. Furthermore, the transgenic plants showed enhanced level of resistance against the virulent fungus Botrytis cinerea. Thus, our data suggest that the jasmonic acid carboxyl methyltransferase is a key enzyme for jasmonate-regulated plant responses. Activation of JMT expression leads to production of methyl jasmonate that could act as an intracellular regulator, a diffusible intercellular signal transducer, and an airborne signal mediating intra- and interplant communications.

Nitric oxide and salicylic acid signaling in plant defense

Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the H2O2-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to IκBα and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-1). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-1). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals.

Ozone Sensitivity in Hybrid Poplar Is Correlated with a Lack of Defense-Gene Activation1

Ozone is a major gaseous pollutant thought to contribute to forest decline. Although the physiological and morphological responses of forest trees to ozone have been well characterized, little is known about the molecular basis for these responses. Our studies compared the response to ozone of ozone-sensitive and ozone-tolerant clones of hybrid poplar (Populus maximowizii × Populus trichocarpa) at the physiological and molecular levels. Gas-exchange analyses demonstrated clear differences between the ozone-sensitive clone 388 and the ozone-tolerant clone 245. Although ozone induced a decrease in photosynthetic rate and stomatal conductance in both clones, the magnitude of the decrease in stomatal conductance was significantly greater in the ozone-tolerant clone. RNA-blot analysis established that ozone-induced mRNA levels for phenylalanine ammonia-lyase, O-methyltransferase, a pathogenesis-related protein, and a wound-inducible gene were significantly higher in the ozone-tolerant than in the ozone-sensitive plants. Wound- and pathogen-induced levels of these mRNAs were also higher in the ozone-tolerant compared with the ozone-sensitive plants. The different physiological and molecular responses to ozone exposure exhibited by clones 245 and 388 suggest that ozone tolerance involves the activation of salicylic-acid- and jasmonic-acid-mediated signaling pathways, which may be important in triggering defense responses against oxidative stress.

Oligogalacturonide defense signals in plants: large fragments interact with the plasma membrane in vitro.

Oligogalacturonides are plant cell wall-derived regulatory molecules which stimulate defense gene expression during pathogenesis. In vitro, these compounds enhance the phosphorylation of an approximately 34-kDa protein (pp34) in purified plasma membranes from potato and tomato leaves. We now show that polygalacturonate-enhanced phosphorylation of pp34 occurs in plasma membranes purified from tomato roots, hypocotyls, and stems and from undifferentiated potato cells. Furthermore, a similar phosphorylation is detected in leaf plasma membranes from soybean, a plant distantly related to tomato. Purified oligogalacturonides 13 to at least 26 residues long stimulate pp34 thiophosphorylation in vitro. This stimulation pattern differs from the induction of many known defense responses in vivo, where a narrower range of smaller fragments, between approximately 10 and 15 residues long, are active. On the basis of these differences we suggest that observed effects of applied exogenous oligogalacturonides on defense responses may not necessarily reflect the situation during pathogenesis. The cell wall could act as a barrier to many exogenous oligo- and polygalacturonides as well as other large regulatory ligands.

Oligopeptide elicitor-mediated defense gene activation in cultured parsley cells.

We have used suspension-cultured parsley cells (Petroselinum crispum) and an oligopeptide elicitor derived from a surface glycoprotein of the phytopathogenic fungus Phytophthora megasperma f.sp. glycinea to study the signaling pathway from elicitor recognition to defense gene activation. Immediately after specific binding of the elicitor by a receptor in the plasma membrane, large and transient increases in several inorganic ion fluxes (Ca2+, H+, K+, Cl-) and H2O2 formation are the first detectable plant cell responses. These are rapidly followed by transient changes in the phosphorylation status of various proteins and by the activation of numerous defense-related genes, concomitant with the inactivation of several other, non-defense-related genes. A great diversity of cis-acting elements and trans-acting factors appears to be involved in elicitor-mediated gene regulation, similar to the apparently complex nature of the signal transduced intracellularly. With few exceptions, all individual defense responses analyzed in fungus-infected parsley leaves have been found to be closely mimicked in elicitor-treated, cultured parsley cells, thus validating the use of the elicitor/cell culture system as a valuable model system for these types of study.