A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance.

Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes. To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS). The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity. We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5. Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA. Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant. Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants. Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.     

Hydrogen peroxide does not function downstream of salicylic acid in the induction of PR protein expression

The roles of salicylic acid (SA) and H₂O₂ in the induction of PR proteins in tobacco have been examined. Studies were conducted on wild-type tobacco and plants engineered to express a bacterial salicylate hydroxylase capable of metabolizing SA to catechol (SH-L plants). Wild-type and PR-1a—GUS-transformed plants express PR-1a following challenge with Pseudomonas syringae pathovar syringae , SA or 2,6-dichloro-isonicotinic acid (INA). In contrast, SH-L plants failed to respond to SA but did express PR-1a following INA treatment. H₂O₂ and the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT) were found to be weak inducers of PR-1a expression (relative to SA) in wild-type tobacco but were unable to induce PR-1a in SH-L plants, suggesting that the action of these compounds depends upon the accumulation of SA. A model has been proposed suggesting that SA binds to and inhibits a catalase inducing an increase in H₂O₂ leading to PR protein expression. Catalase activity has been measured in tobacco and no significant changes in activity following infection with P. syringae pv. syringae were detected. Furthermore, inhibition of catalase activity in vitro in plant extracts requires pre-incubation and only occurs at SA concentrations above 250 µM. Leaf disks pre-incubated with 1 mM SA do accumulate SA to these levels and PR-1a is efficiently induced but there is no apparent inhibition of catalase activity. It is also shown that a SA-responsive gene, PR-1a, and a H₂O₂-sensitive gene, AoPR-1, are both relatively insensitive to 3-AT suggesting that induction of these genes is unlikely to be due entirely to inhibition of an endogenous catalase.     

Extracellular pyridine nucleotides induce PR gene expression and disease resistance in Arabidopsis

Although it is well known that the pyridine nucleotides NAD and NADP function inside the cell to regulate intracellular signaling processes, recent evidence from animal studies suggests that NAD(P) also functions in the extracellular compartment (ECC). Extracellular NAD(P) [eNAD(P)] can either directly bind to plasma membrane receptors or be metabolized by ecto-enzymes to produce cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, and/or may ADP-ribosylate cell-surface receptors, resulting in activation of transmembrane signaling. In this study, we report that, in plants, exogenous NAD(P) induces the expression of pathogenesis-related ( PR ) genes and resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Chelation of Ca²⁺ by EGTA significantly inhibits the induction of PR genes by exogenous NAD(P), suggesting that exogenous NAD(P) may induce PR genes through a pathway that involves Ca²⁺ signaling. We show that exogenous application of NAD(P) causes accumulation of the defense signal molecule salicylic acid (SA), and induces both SA/NPR1-dependent and -independent PR gene expression and disease resistance. Furthermore, we demonstrate that NAD(P) leaks into the plant ECC after mechanical wounding and pathogen infection, and that the amount of NAD(P) leaking into the ECC after P. syringae pv. tobacco DC3000/ avrRpt2 infection is sufficient for induction of both PR gene expression and disease resistance. We propose that NAD(P) leakage from cells losing membrane integrity upon environmental stress may function as an elicitor to activate plant defense responses. Our data provide evidence that eNAD(P) functions in plant signaling, and illustrate the potential importance of eNAD(P) in plant innate immunity.     

Protein dephosphorylation mediates salicylic acid-induced expression of PR-1 genes in tobacco

Several lines of evidence suggest that salicylic acid (SA) is an endogenous signal for the activation of several plant defense responses, including the expression of genes encoding pathogenesis-related (PR) proteins such as the acidic PR-1 proteins. During recent years, studies have suggested that interaction of SA with catalase and ascorbate peroxidase leads to two signals in tobacco - elevated H₂O₂ levels and lipid peroxides. However, to date, relatively little is known about the molecular and biochemical mechanisms that mediate transduction beyond these signals or through other SA-effector proteins. Using protein kinase and phosphatase inhibitors, this study demonstrates that PR-1 gene induction can be mediated by dephosphorylation of serine/threonine residue(s) of two or more unidentified phosphoproteins. The protein phosphatase inhibitors, okadaic acid and calyculin A blocked SA-mediated induction of PR-1 genes, implying the involvement of a phosphoprotein downstream of SA. The protein kinase inhibitors K-252a and staurosporine induced PR-1 gene expression. PR-1 gene induction by K-252a was suppressed by okadaic acid. Surprisingly, this induction was also suppressed in NahG transgenic tobacco plants which convert SA to catechol. Moreover, K-252a stimulated production of SA and its glucoside, suggesting that another phosphoprotein acts upstream of SA. Taken together, these results suggest that there are two (or more) phosphoproteins which function in the same signal transduction pathway leading to PR-1 gene induction. The SA-inducible acidic PR-2 genes were similarly affected by the inhibitors, while the genes for actin and phenylalanine ammonia lyase were not.     

The leucine-rich repeat (LRR) protein, CaLRR1, interacts with the hypersensitive induced reaction (HIR) protein, CaHIR1, and suppresses cell death induced by the CaHIR1 protein

Leucine-rich repeat proteins (LRRs) function in a number of signal transduction pathways via protein–protein interactions. The gene encoding a small protein of pepper, CaLRR1 , is specifically induced upon pathogen challenge and treatment with pathogen-associated molecular patterns (PAMPs). We identified a pepper hypersensitive induced reaction (CaHIR1) protein that interacts with the LRR domain of the CaLRR1 protein using yeast two-hybrid screening. Ectopic expression of the pepper CaHIR1 gene induces cell death in tobacco and Arabidopsis, indicating that the CaHIR1 protein may be a positive regulator of HR-like cell death. Because transformation is very difficult in pepper plants, we over-expressed CaLRR1 and CaHIR1 in Arabidopsis to determine cellular functions of the two genes. The over-expression of the CaHIR1 gene, but not the CaLRR1 gene, in transgenic Arabidopsis confers disease resistance in response to Pseudomonas syringae infection, accompanied by the strong expression of PR genes, the accumulation of both salicylic acid and H₂O₂, and K⁺ efflux in plant cells. In Arabidopsis and tobacco plants over-expressing both CaHIR1 and CaLRR1 , the CaLRR1 protein suppresses not only CaHIR1 -induced cell death, but also PR gene expression elicited by CaHIR1 via its association with HIR protein. We propose that the CaLRR1 protein functions as a novel negative regulator of CaHIR1-mediated cell death responses in plants.     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.     

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin α₂β₁. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin α₂β₁ and affects the biological outcome of host–pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the α₂β₁ integrin, because (i) both adherence and internalization of the scl1- inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-α₂ integrin-subunit antibody and type I collagen, (iii) recombinant α₂-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the α₂β₁ integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.     

Studies on the mechanism of resistance to Bipolaris sorokiniana in the barley lesion mimic mutant bst1

The Bipolaris sorokiniana tolerant 1 ( bst1 ) barley mutant is derived from fast neutron-irradiated seeds of wild-type Bowman( Rph3 ). The induced mutation was genetically localized to a position on chromosome 5HL distal to the centromere using amplified fragment length polymorphism markers. In addition, the defence responses and related gene expression in the bst1 mutant after fungal challenge were compared with those occurring in wild-type plants. Hydrogen peroxide generation, determined by 3,3-diaminobenzidine staining, revealed a clearly reduced level of bst1 , compared with the wild-type, during the entire experimental time: 8–120 h post-inoculation (hpi). At 48 hpi, the wild-type samples displayed twice as much fungal mass and three times greater H₂O₂ production than bst1 . At the same time, staining of B. sorokiniana showed less fungal growth in the spontaneous lesions of bst1 compared with the wild-type. Monitoring of defence-related genes at 48 hpi demonstrated strong expression of PR-1a , PR-2 , PR-5 and PR-10 in bst1. A gene coding for a unique oxidoreductase enzyme, designated as HCP1 , was expressed at much higher levels in inoculated leaves of the bst1 mutant than in those of the wild-type plant. Taken together, the results suggest that the defence to B. sorokiniana largely relies on salicylic acid-responsive pathogenesis-related ( PR ) genes, as well as selected reactive oxygen species and unknown HCP1 -associated factors.     

Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels

Numerous studies argue that salicylic acid (SA) is an important component of the plant signal transduction pathway(s) leading to disease resistance. The discovery that the SA-binding protein is a catalase, whose activity is blocked by SA, led to the proposal that one of SA's modes of action is to inhibit this H₂O₂-degrading enzyme and thus elevate H₂O₂ levels. To test this model, an attempt was made to mimic the action of SA by reducing the synthesis of catalase using antisense RNA technology. Analyses of transgenic tobacco plants that expressed the tobacco catalase 1 ( cat1 ) or catalase 2 ( cat2 ) gene in an antisense orientation indicate that there is no correlation between modest to high levels of reduction in catalase activity and activation of plant defenses such as pathogenesis-related (PR)-1 protein synthesis. However, three independent antisense catalase transgenic plants (ASCAT1 Nos 16, 17, and 28), which exhibited the most severe reduction in catalase activity (∼90% or more), developed chlorosis or necrosis on some of their lower leaves. These same leaves accumulated very high levels of PR-1 proteins and showed enhanced resistance to tobacco mosaic virus. Necrosis and elevated SA, which appear to result from severe depression of catalase levels, may be responsible for the induction of these defense responses.     

An important role of a BAHD acyl transferase-like protein in plant innate immunity

Salicylic acid (SA) is an important regulator of plant resistance to biotrophic and hemi-biotrophic pathogens. The enhanced pseudomonas susceptibility 1 ( eps1 ) mutant in Arabidopsis thaliana is hypersusceptible to both virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae . Through positional cloning, the EPS1 gene was isolated and found to encode a novel member of the BAHD acyltransferase superfamily. Pathogen-induced accumulation of SA and expression of pathogenesis-related ( PR ) genes were compromised in the eps1 mutant. SA could induce PR1 gene expression and restore disease resistance in the eps1 mutant. These results suggest that EPS1 functions upstream of SA and may be involved directly in synthesis of a precursor or a regulatory molecule for SA biosynthesis. Mutations of EPS1 or other genes important for SA accumulation or signaling conferred enhanced resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola in the Nossen-0 background but had little effect in the Columbia-0 background. These results suggest that there is natural variation among Arabidopsis ecotypes with respect to the antagonistic cross-talk between defense signaling pathways against various types of microbial pathogens.     

Expression profiles of two novel lipoxygenase genes in Populus deltoides

In the present study, we cloned two lipoxygenase genes, PdLOX1 and PdLOX2 (GenBank accession no. DQ131178, DQ131179), from Populus deltoides cv. ‘Lux’ (I-69/55). A prokaryotic expression analysis of PdLOX1 and PdLOX2 revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the expected lipoxygenase activities. Chromatogram analysis indicated that the two lipoxygenase mainly possess 13-LOX activity. Phylogenetic analysis of the derived amino acid sequences of known lipoxygenases revealed that PdLOX1 and PdLOX2 were members of the type 2 13-LOX family of genes. This class of lipoxygenases is known to be involved in biotic and abiotic stress. Using real-time RT-PCR, we evaluated PdLOX1 and PdLOX2 expression following exposure to a Poplar fungal pathogen (Marssonina brunnea f. sp. Multigermtubi), mechanical injury, methyl jasmonate (MeJA), or salicylic acid (SA). We report that both PdLOX1 and PdLOX2 expression levels were increased following exposure to M. brunnea f. sp. Multigermtubi, with the pathogen exerting a relatively stronger influence on PdLOX1 expression. Furthermore, expression levels of the two genes were also up-regulated by mechanical damage and exposure to MeJA. In contrast, both PdLOX1 and PdLOX2 expression was down-regulated by SA treatment. We propose that the two novel lipoxygenases may play an important role in Poplar resistance to biotic and abiotic stress.     

Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana

Nitric oxide (NO) is involved together with reactive oxygen species (ROS) in the activation of various stress responses in plants. We have used ozone (O₃) as a tool to elicit ROS-activated stress responses, and to activate cell death in plant leaves. Here, we have investigated the roles and interactions of ROS and NO in the induction and regulation of O₃-induced cell death. Treatment with O₃ induced a rapid accumulation of NO, which started from guard cells, spread to adjacent epidermal cells and eventually moved to mesophyll cells. During the later time points, NO production coincided with the formation of hypersensitive response (HR)-like lesions. The NO donor sodium nitroprusside (SNP) and O₃ individually induced a large set of defence-related genes; however, in a combined treatment SNP attenuated the O₃ induction of salicylic acid (SA) biosynthesis and other defence-related genes. Consistent with this, SNP treatment also decreased O₃-induced SA accumulation. The O₃-sensitive mutant rcd1 was found to be an NO overproducer; in contrast, Atnoa1/rif1 ( Arabidopsis nitric oxide associated 1/resistant to inhibition by FSM1 ), a mutant with decreased production of NO, was also O₃ sensitive. This, together with experiments combining O₃ and the NO donor SNP suggested that NO can modify signalling, hormone biosynthesis and gene expression in plants during O₃ exposure, and that a functional NO production is needed for a proper O₃ response. In summary, NO is an important signalling molecule in the response to O₃.