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.     

Tissue Variance of Salicylic Acid-sensitive Catalase and Enhancement of Disease Resistance with Exogenous Salicylic Acid in Maize

The response to pathogen mediated by salicylic acid (SA) in plant required not only a high level of SA but also an effective SA signal perception and transduction mechanism. The maize ( Zea mays L. ) leaves contained extremely low level of free SA. Catalases from different maize tissues also exhibited different sensitivity to SA. Catalases from leaves were sensitive to SA, but roots contained SA-insensitive eatalases, indicating the presence of different tissue specific catalase isozymes. It seems that there is such an effective mechanism signal sensitization and signal conductance in maize leaves just the same as in tabacco and Arabidopsis. The ability of resistance to Helminthosporium turcicum Pass. in maize leaves can be enhanced by SA.     

Expression of tobacco class II catalase gene activates the endogenous homologous gene and is associated with disease resistance in transgenic potato plants

We have previously shown that healthy potato plants respond poorly to salicylic acid (SA) for activating disease resistance against the late blight fungal pathogen Phytophthora infestans. However, SA is essential for the establishment of potato systemic acquired resistance (SAR) against P. infestans after treatment with the fungal elicitor arachidonic acid (AA). To understand the molecular mechanisms through which AA induces SA-dependent SAR in potato, we have recently studied the expression of potato class II catalase (Cat2St) in comparison with its tobacco homologue, Cat2Nt, which has previously been shown to bind SA. In the present study, we show that tobacco Cat2Nt is expressed at high levels and accounts for almost half of total SA-binding activity detected in tobacco leaves. In contrast, potato Cat2St is not expressed in healthy leaves, which is associated with the low SA responsiveness of potato plants for activation of disease resistance mechanisms. Upon treatment with AA, expression of potato Cat2St is induced not only in AA-treated leaves, but also in the upper untreated parts of the plants, concomitant with the establishment of SA -dependent SAR to P. infestans. Moreover, expression of the tobacco Cat2Nt gene in transgenic potato plants leads to constitutive expression of the endogenous potato Cat2St gene and is associated with enhanced resistance to P. infestans. These results collectively indicate that plant SA-binding class II catalases may play an important role in the development of disease resistance, possibly by serving as biological targets of SA.     

Proteomic analysis on salicylic acid-induced salt tolerance in common wheat seedlings (Triticum aestivum L.)

The influence of salicylic acid (SA) on the salt tolerance mechanism in seedlings of common wheat (Triticum aestivum L.) was investigated using physiological measurements combined with global expression profiling (proteomics). In the present study, 0.5mM SA significantly reduced NaCl-induced growth inhibition in wheat seedlings, manifesting as increased fresh weights, dry weights, and photosynthetic pigments, but decreased lipid peroxidation. Two-week-old wheat seedlings treated with 0.5mM SA, 250mM NaCl and 250mM NaCl+0.5mM SA for 3days were used for the proteomic analyses. In total, 39 proteins differentially regulated by both salt and SA were revealed by 2D PAGE, and 38 proteins were identified by MALDI-TOF/TOF MS. The identified proteins were involved in various cellular responses and metabolic processes including signal transduction, stress defense, energy, metabolism, photosynthesis, and others of unknown function. All protein spots involved in signal transduction and the defense response were significantly upregulated by SA under salt stress, suggesting that these proteins could play a role in the SA-induced salt resistance in wheat seedlings.     

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.     

Systemic acquired resistance signal transduction

Systemic acquired resistance (SAR) is an inducible plant defense response in which a prior foliar pathogen infection activates resistance in noninfected foliar tissues. Salicylic acid (SA) accumulation is essential for the establishment of SAR. While SA is probably not the long‐distance systemic signal instrumental for SAR activation, it is required for transduction of the signal in noninfected tissues. Although SAR was first described as a response to necrogenic pathogen infection, synthetic chemicals have been identified that effectively activate SAR. Elucidation of SAR signal transduction has been facilitated by the identification and characterization of Arabidopsis mutants. Disease lesion mimic mutants exhibit constitutive SAR as well as spontaneous lesion formation similar to pathogen‐associated hypersensitive cell death. Some disease lesion mimic mutants do not exhibit a lesioned phenotype when SA accumulation is prevented, thereby providing evidence for a feedback loop in SAR signal transduction. Moreover, characterization of mutants compromised for SAR activation has provided additional evidence for common signaling components between SAR and gene‐for‐gene resistance.     

Proteomic analysis of salicylic acid induced resistance to Mungbean Yellow Mosaic India Virus in Vigna mungo

The role of salicylic acid (SA) in inducing resistance to MYMIV infection in Vigna mungo has been elucidated by proteomics. Twenty-nine proteins identified by MALDI-TOF/TOF, predicted to be involved in stress responses, metabolism, photosynthesis, transport and signal transduction, showed increased abundance upon SA treatment. Susceptible plants showed characteristic yellow mosaic symptoms upon MYMIV infection. A concentration dependent decrease in physiological symptoms associated with MYMIV was observed upon exogenous SA treatment prior to viral inoculation; and no visible symptom was observed at 100 μM SA. SA treatment stimulated SOD and GPX activity and inhibited CAT activity thus preventing ROS mediated damage. Significant increase in chlorophyll, protein, carbohydrate, phenolic content and H(2)O(2) were observed. Involvement of calmodulin for transmission of defense signal by SA is suggested. A metabolic reprogramming leading to enhanced synthesis of proteins involved in primary and secondary metabolisms is necessary for SA mediated resistance to MYMIV. Identification of proteins showing increased abundance, involved in photosynthetic process is a significant finding which restores virus-induced degradation of the photosynthetic apparatus and provides enhanced metabolites required for repartition of resources towards defense.     

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8.     

Constitutive salicylic acid-dependent signaling in cpr1 and cpr6 mutants requires PAD4

Salicylic acid (SA)-dependent signaling controls activation of a set of plant defense mechanisms that are important for resistance to a variety of microbial pathogens. Many Arabidopsis mutants that display altered SA-dependent signaling have been isolated. We used double mutant analysis to determine the relative positions of the pad4, cpr1, cpr5, cpr6, dnd1 and dnd2 mutations in the signal transduction network leading to SA-dependent activation of defense gene expression and disease resistance. The pad4 mutation causes failure of SA accumulation in response to infection by certain pathogens, while the other mutations cause constitutively high levels of SA, defense gene expression and resistance. The cpr1 pad4, cpr5 pad4, cpr6 pad4, dnd1 pad4 and dnd2 pad4 double mutants were constructed and assayed for stature, presence of spontaneous lesions, resistance to Pseudomonas syringae and Peronospora parasitica, SA levels, expression of PAD4, PR-1 and PDF1.2, and accumulation of camalexin. We found that the effects of the cpr1 and cpr6 mutations on SA-dependent gene expression are completely dependent on PAD4 function. In contrast, SA accumulation in the lesion-mimic mutant cpr5 is partially PAD4-independent, while in dnd1 and dnd2 mutants it is completely PAD4-independent. A model describing a possible arrangement of activities in the signal transduction network is presented.     

Induction of pathogen resistance and pathogenesis-related genes in tobacco by a heat-stable Trichoderma mycelial extract and plant signal messengers

Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings ( Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica . The nonpathogenic mycelial extract induced expression of PR-1b and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.     

Characterization of the Interaction between the Cohesin Subunits Rad21 and SA1/2

The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the ''core'' cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301–750 aa), Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT) and middle part (MP) SA-binding motif, located at 60–81 aa of the N-terminus and 383–392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, α-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L³⁸⁵, F³⁸⁹, and T³⁹⁰) in this α-helical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SA-binding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21.     

Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid

Damage-associated molecular pattern molecules (DAMPs) signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3) is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i) MAPK activation, ii) defense-related gene expression, iii) callose deposition, and iv) enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast). Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA), which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.     

植物系统获得的抗病性和信号传导

植物在长期的进化过程中,需要不断地抵抗病原微生物的侵害。在这种长期相互影响的共进化过程中,植物逐渐形成一系列复杂而行之有效的保护机制来抵御病原微生物的侵染。在植物抵御病原微生物侵染的过程中,宿主植物的抗病基因（R）产物与病原微生物无毒基因（Avr）产物的...     

植物抗逆性与水杨酸介导的信号传导途径的关系

基因表达既受发育过程的调控又受外界环境的影响。无论是内因还是外因诱发一组基因表达时都涉及信号传导(signal transduction)问题。局部器官和组织所发生的生理变化的信息要传递到远处的组织,引起基因表达时间和空间上的协调。信号传导途径的研究是当今分子生物学的研究热点之一。 作为信号传递的分子主要是小分子物质,属于次生代谢产物。也发现某些小肽具有信号分子的功能。信号分子可以在胞间扩散,亦可通过输导组织传送到远处的器官。近年来研究甚多的一种信号     

Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.     

Ozone sensitivity in hybrid poplar correlates with insensitivity to both salicylic acid and jasmonic acid. The role of programmed cell death in lesion formation

Our earlier studies demonstrated that the ozone-sensitive hybrid poplar clone NE-388 displays an attenuated level of ozone-, wound-, and phytopathogen-induced defense gene expression. To determine if this reduced gene activation involves signal transduction pathways dependent on salicylic acid (SA) and/or jasmonic acid (JA), we compared the responses of NE-388 and an ozone-tolerant clone, NE-245, to these signal molecules. JA levels increased in both clones in response to ozone, but only minimal increases in SA levels were measured for either clone. Treatment with SA and methyl jasmonate induced defense gene expression only in NE-245, indicating that NE-388 is insensitive to these signal molecules. DNA fragmentation, an indicator of programmed cell death (PCD), was detected in NE-245 treated with either ozone or an avirulent phytopathogen, but was not detected in NE-388. We conclude that these clones undergo two distinct mechanisms of ozone-induced lesion formation. In NE-388, lesions appear to be due to toxic cell death resulting from a limited ability to perceive and subsequently activate SA- and/or JA-mediated antioxidant defense responses. In NE-245, SA-dependent PCD precedes lesion formation via a process related to the PCD pathway activated by phytopathogenic bacteria. These results support the hypothesis that ozone triggers a hypersensitive response.     

Salicylic Acid Interferes with Tobacco Mosaic Virus Replication via a Novel Salicylhydroxamic Acid-Sensitive Mechanism

Salicylic acid (SA) induces resistance to all plant pathogens, including bacteria, fungi, and viruses, but the mechanism by which SA engenders resistance to viruses is not known. Pretreatment of tobacco mosaic virus (TMV)-susceptible (nn genotype) tobacco tissue with SA reduced the levels of viral RNAs and viral coat protein accumulating after inoculation with TMV. Viral RNAs were not affected equally, suggesting that SA treatment interferes with TMV replication. Salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial alternative oxidase, antagonized both SA-induced resistance to TMV in nn genotype plants and SA-induced acquired resistance in resistant (NN genotype) tobacco. SHAM did not inhibit induction of the PR-1 pathogenesis-related protein or induction of resistance to Erwinia carotovora or Botrytis cinerea by SA. This indicates that SA induces resistance to TMV via a novel SHAM-sensitive signal transduction pathway (potentially involving alternative oxidase), which is distinct from that leading to resistance to bacteria and fungi.     

ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response

The previously reported Arabidopsis dominant gain-of-function mutant accelerated cell death6-1 (acd6-1) shows spontaneous cell death and increased disease resistance. acd6-1 also confers increased responsiveness to the major defense signal salicylic acid (SA). To further explore the role of ACD6 in the defense response, we cloned and characterized the gene. ACD6 encodes a novel protein with putative ankyrin and transmembrane regions. It is a member of one of the largest uncharacterized gene families in higher plants. Steady state basal expression of ACD6 mRNA required light, SA, and an intact SA signaling pathway. Additionally, ACD6 mRNA levels were increased in the systemic, uninfected tissue of Pseudomonas syringae-infected plants as well as in plants treated with the SA agonist benzothiazole (BTH). A newly isolated ACD6 loss-of-function mutant was less responsive to BTH and upon P. syringae infection had reduced SA levels and increased susceptibility. Conversely, plants overexpressing ACD6 showed modestly increased SA levels, increased resistance to P. syringae, and BTH-inducible and/or a low level of spontaneous cell death. Thus, ACD6 is a necessary and dose-dependent activator of the defense response against virulent bacteria and can activate SA-dependent cell death.