A role for salicylic acid and NPR1 in regulating cell growth in Arabidopsis

Salicylic acid (SA) plays a key role in activating defenses and cell death during plant-pathogen interactions. In response to some pathogens, SA also limits the extent of cell death, indicating that it acts positively or negatively depending on the host-pathogen interaction. In addition, we previously showed that SA affects cell growth in the Arabidopsis defense-related mutants accelerated cell death 6-1 (acd6-1) and aberrant growth and death 2 (agd2). Using acd6-1, agd2 and two other defense-related mutants, lesion simulating disease 6 (lsd6), suppressor of SA-insensitivity (ssi1), we show here in detail that SA regulates cell growth by specifically affecting cell enlargement, endoreduplication and/or cell division. We find that SA can act either positively or negatively to regulate cell growth depending on the context in which signaling occurs. Additionally, Nonexpressor of PR 1 (NPR1), a key SA signaling protein important for regulating defenses and cell death, also acts to promote cell division and/or suppress endoreduplication during leaf development. We propose that SA interacts with multiple receptors or signaling pathways to control cellular alterations during normal development, pathogen attack and/or stress situations. We suggest that SA and NPR1 play broader roles in cell fate control than has previously been understood.     

Genetic dissection of salicylic acid-mediated defense signaling networks in Arabidopsis

Properly coordinated defense signaling networks are critical for the fitness of plants. One hub of the defense networks is centered on salicylic acid (SA), which plays a key role in activating disease resistance in plants. However, while a number of genes are known to affect SA-mediated defense, relatively little is known about how these gene interact genetically with each other. Here we exploited the unique defense-sensitized Arabidopsis mutant accelerated cell death (acd) 6-1 to dissect functional relationships among key components in the SA hub. We show that while enhanced disease susceptibility (eds) 1-2 and phytoalexin deficient (pad) 4-1 suppressed acd6-1-conferred small size, cell death, and defense phenotypes, a combination of these two mutations did not incur additive suppression. This suggests that EDS1 and PAD4 act in the same signaling pathway. To further evaluate genetic interactions among SA regulators, we constructed 10 pairwise crosses in the acd6-1 background among mutants defective in: SA INDUCTION-DEFICIENT 2 for SA biosynthesis; AGD2-LIKE DEFENSE 1, EDS5, and PAD4 for SA accumulation; and NONEXPRESSOR OF PR GENES 1 for SA signaling. Systematic analysis of the triple mutants based on their suppression of acd6-1-conferred phenotypes revealed complex and interactive genetic relationships among the tested SA genes. Our results suggest a more comprehensive view of the gene networks governing SA function and provide a framework for further interrogation of the important roles of SA and possibly other signaling molecules in regulating plant disease resistance.     

Genetic analysis of acd6-1 reveals complex defense networks and leads to identification of novel defense genes in Arabidopsis

Pathogen infection leads to the activation of defense signaling networks in plants. To study these networks and the relationships between their components, we introduced various defense mutations into acd6-1 , a constitutive gain-of-function Arabidopsis mutant that is highly disease resistant. acd6-1 plants show spontaneous cell death, reduced stature, and accumulate high levels of camalexin (an anti-fungal compound) and salicylic acid (SA; a signaling molecule). Disruption of several defense genes revealed that in acd6-1 , SA levels/signaling were positively correlated with the degree of disease resistance and defense gene expression. Salicylic acid also modulates the severity of cell death. However, accumulation of camalexin in acd6-1 is largely unaffected by reducing the level of SA. In addition, acd6-1 shows ethylene- and jasmonic acid-mediated signaling that is antagonized and therefore masked by the presence of SA. Mutant analysis revealed a new relationship between the signaling components NPR1 and PAD4 and also indicated that multiple defense pathways were required for phenotypes conferred by acd6-1 . In addition, our data confirmed that the size of acd6-1 was inversely correlated with SA levels/signaling. We exploited this unique feature of acd6-1 to identify two genes disrupted in acd6-1 suppressor ( sup ) mutants: one encodes a known SA biosynthetic component (SID2) and the other encodes an uncharacterized putative metalloprotease (At5g20660). Taken together, acd6-1 is a powerful tool not only for dissecting defense regulatory networks but also for discovering novel defense genes.     

Arabidopsis mutants compromised for the control of cellular damage during pathogenesis and aging

Mutants of Arabidopsis thaliana which exhibit accelerated cell death in response to pathogens were isolated and characterized to gain insight into how symptom severity and disease resistance are modulated. This paper describes mutants that fall into one of two complementation groups that were identified. A novel feature of these mutants is that they are unable to control the rate and extent of cell death after exposure to a variety of stimuli that induce senescence responses. Thus, accelerated cell death ( acd1 ) mutants show rapid, spreading necrotic responses to both virulent and avirulent Pseudomonas syringae pv. maculicola or pv. tomato pathogens and to ethylene. In addition, they develop necrotic lesions as they age and are sensitive to mechanical stress in a developmentally controlled manner. The acd1 mutants are also susceptible to opportunistic pathogens and show decreased growth inhibition of a heterologous pathogen of bean. The signal for lesion formation is not necessarily due to pathogens or wounding since plants grown aseptically also develop necrotic lesions. The lesions formed under a variety of conditions resemble those produced during a pathogen-induced rapid cell death response (the hypersensitive response, HR). Analysis of these acd1 mutants may help to explain the molecular basis of the HR and the relationship between this response and the normal process of senescence.     

The role of salicylic acid in the induction of cell death in Arabidopsis acd11

Salicylic acid (SA) is implicated in the induction of programmed cell death (PCD) associated with pathogen defense responses because SA levels increase in response to PCD-inducing infections, and PCD development can be inhibited by expression of salicylate hydroxylase encoded by the bacterial nahG gene. The acd11 mutant of Arabidopsis (Arabidopsis thaliana L. Heynh.) activates PCD and defense responses that are fully suppressed by nahG. To further study the role of SA in PCD induction, we compared phenotypes of acd11/nahG with those of acd11/eds5-1 and acd11/sid2-2 mutants deficient in a putative transporter and isochorismate synthase required for SA biosynthesis. We show that sid2-2 fully suppresses SA accumulation and cell death in acd11, although growth inhibition and premature leaf chlorosis still occur. In addition, application of exogenous SA to acd11/sid2-2 is insufficient to restore cell death. This indicates that isochorismate-derived compounds other than SA are required for induction of PCD in acd11 and that some acd11 phenotypes require NahG-degradable compounds not synthesized via isochorismate.     

Fumonisin B1-induced cell death in arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways

We have established an Arabidopsis protoplast model system to study plant cell death signaling. The fungal toxin fumonisin B1 (FB1) induces apoptosis-like programmed cell death (PCD) in wild-type protoplasts. FB1, however, only marginally affects the viability of protoplasts isolated from transgenic NahG plants, in which salicylic acid (SA) is metabolically degraded; from pad4-1 mutant plants, in which an SA amplification mechanism is thought to be impaired; or from jar1-1 or etr1-1 mutant plants, which are insensitive to jasmonate (JA) or ethylene (ET), respectively. FB1 susceptibility of wild-type protoplasts decreases in the dark, as does the cellular content of phenylalanine ammonia-lyase, a light-inducible enzyme involved in SA biosynthesis. Interestingly, however, FB1-induced PCD does not require the SA signal transmitter NPR1, given that npr1-1 protoplasts display wild-type FB1 susceptibility. Arabidopsis cpr1-1, cpr6-1, and acd2-2 protoplasts, in which the SA signaling pathway is constitutively activated, exhibit increased susceptibility to FB1. The cpr6-1 and acd2-2 mutants also constitutively express the JA and ET signaling pathways, but only the acd2-2 protoplasts undergo PCD in the absence of FB1. These results demonstrate that FB1 killing of Arabidopsis is light dependent and requires SA-, JA-, and ET-mediated signaling pathways as well as one or more unidentified factors activated by FB1 and the acd2-2 mutation.     

The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth.

We isolated a dominant gain-of-function Arabidopsis mutant, accelerated cell death 6 (acd6), with elevated defenses, patches of dead and enlarged cells, reduced stature, and increased resistance to Pseudomonas syringae. The acd6-conferred phenotypes are suppressed by removing a key signaling molecule, salicylic acid (SA), by using the nahG transgene, which encodes SA hydroxylase. This suppression includes phenotypes that are not induced by application of SA to wild-type plants, indicating that SA acts with a second signal to cause many acd6-conferred phenotypes. acd6-nahG plants show hyperactivation of all acd6-conferred phenotypes after treatment with a synthetic inducer of the SA pathway, benzo(1,2, 3)thiadiazole-7-carbothioic acid (BTH), suggesting that SA acts with and also modulates the levels and/or activity of the second defense signal. acd6 acts partially through a NONEXPRESSOR OF PR 1 (NPR1) gene-independent pathway that activates defenses and confers resistance to P. syringae. Surprisingly, BTH-treated acd6-nahG plants develop many tumor-like abnormal growths, indicating a possible role for SA in modulating cell growth.     

Characterisation of an Arabidopsis-Leptosphaeria maculans pathosystem: resistance partially requires camalexin biosynthesis and is independent of salicylic acid, ethylene and jasmonic acid signalling

Out of 168 Arabidopsis accessions screened with isolates of Leptosphaeria maculans, one (An-1) showed clear disease symptoms. In order to identify additional components involved in containment of L. maculans in Arabidopsis, a screen for L. maculans-susceptible (lms) mutants was performed. Eleven lms mutants were isolated, which displayed differential susceptibility responses to L. maculans. lms1 was crossed with Columbia (Col-0) and Ws-0, and mapping data for both populations showed the highest linkage to a region on chromosome 2. Reduced levels of PR-1 and PDF1.2 expression were found in lms1 compared to wild-type plants 48 h after pathogen inoculation. In contrast, the lms1 mutant displayed upregulation of either marker gene upon chemical treatment, possibly as an effect of an altered ethylene (ET) response. To assess the contribution of different defence pathways, genotypes implicated in salicylic acid (SA) signalling plants expressing the bacterial salicylate hydroxylase (nahG) gene, non-expressor of PR1 (npr1)-1 and phytoalexin-deficient (pad4-1), jasmonic acid (JA) signalling (coronatine insensitive (coi)1-16, enhanced disease susceptibility (eds)8-1 and jasmonic acid resistant (jar)1-1) and ET signalling (eds4-1, ethylene insensitive (ein)2, ein3-1 and ethylene resistant (etr)1-1) were screened. All the genotypes screened were as resistant as wild-type plants, demonstrating the dispensability of the pathways in L. maculans resistance. When mutants implicated in cell death responses were assayed, responsive to antagonist 1 (ran1)-1 exhibited a weak susceptible phenotype, whereas accelerated cell death (acd)1-20 showed a rapid lesion development. Camalexin is only partially responsible for L. maculans containment in Arabidopsis, as pad3-1 and enhanced susceptibility to Alternaria (esa)1 clearly showed a susceptible response while wild-type levels of camalexin were present in An-1 and lms1. The data presented point to the existence of multiple defence mechanisms controlling the containment of L. maculans in Arabidopsis.     

Positive and negative regulation of salicylic acid-dependent cell death and pathogen resistance in Arabidopsis lsd6 and ssi1 mutants

Salicylic acid (SA) is a key defense molecule in higher plants that is required for resistance to diverse pathogens. A number of mutants of Arabidopsis with elevated resistance to pathogens and constitutive activation of defense-related genes and cell death have been shown to require SA for all of their phenotypes. These mutants potentially identify interesting regulatory genes that control diverse SA responses. When dominant mutations confer SA-dependent phenotypes, it is important to know the genetic basis of dominance in order to draw conclusions on the possible mechanisms of action of the genes identified. Here I characterize the basis of the dominant phenotypes conferred by the ssi1 and lsd6 mutations. I show that ssi1 is haploinsufficient, while lsd6 is a gain-of-function mutation. Thus, SA-dependent responses are under both negative and positive regulation.     

The immune components ENHANCED DISEASE SUSCEPTIBILITY 1 and PHYTOALEXIN DEFICIENT 4 are required for cell death caused by overaccumulation of ceramides in Arabidopsis

Sphingolipids have key functions in plant membrane structure and signaling. Perturbations of plant sphingolipid metabolism often induce cell death and salicylic acid (SA) accumulation; SA accumulation, in turn, promotes sphingolipid metabolism and further cell death. However, the underlying molecular mechanisms remain unclear. Here, we show that the Arabidopsis thaliana lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and its partner PHYTOALEXIN DEFICIENT 4 (PAD4) participate in sphingolipid metabolism and associated cell death. The accelerated cell death 5 (acd5) mutants accumulate ceramides due to a defect in ceramide kinase and show spontaneous cell death. Loss of function of EDS1, PAD4 or SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) in the acd5 background suppressed the acd5 cell death phenotype and prevented ceramide accumulation. Treatment with the SA analogue benzothiadiazole partially restored sphingolipid accumulation in the acd5 pad4 and acd5 eds1 double mutants, showing that the inhibitory effect of the pad4-1 and eds1-2 mutations on acd5-conferred sphingolipid accumulation partly depends on SA. Moreover, the pad4-1 and eds1-2 mutations substantially rescued the susceptibility of the acd5 mutant to Botrytis cinerea. Consistent with this, B. cinerea-induced ceramide accumulation requires PAD4 or EDS1. Finally, examination of plants overexpressing the ceramide synthase gene LAG1 HOMOLOGUE2 suggested that EDS1, PAD4 and SA are involved in long-chain ceramide metabolism and ceramide-associated cell death. Collectively, our observations reveal that EDS1 and PAD4 mediate ceramide (especially long-chain ceramide) metabolism and associated cell death, by SA-dependent and SA-independent pathways.     

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.     

Ozone-induced ethylene production is dependent on salicylic acid,and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death

Ethylene is known to influence plant defense responses including cell death in response to both biotic and abiotic stress factors. However, whether ethylene acts alone or in conjunction with other signaling pathways is not clearly understood. Ethylene overproducer mutants, eto1 and eto3, produced high levels of ethylene and developed necrotic lesions in response to an acute O3 exposure that does not induce lesions in O3-tolerant wild-type Col-0 plants. Treatment of plants with ethylene inhibitors completely blocked O3-induced ethylene production and partially attenuated O3-induced cell death. Analyses of the responses of molecular markers of specific signaling pathways indicated a relationship between salicylic acid (SA)- and ethylene-signaling pathways and O3 sensitivity. Both eto1 and eto3 plants constitutively accumulated threefold higher levels of total SA and exhibited a rapid increase in free SA and ethylene levels prior to lesion formation in response to O3 exposure. SA pre-treatments increased O3 sensitivity of Col-0, suggesting that constitutive high SA levels prime leaf tissue to exhibit increased magnitude of O3-induced cell death. NahG and npr1 plants compromised in SA signaling failed to produce ethylene in response to O3 and other stress factors suggesting that SA is required for stress-induced ethylene production. Furthermore, NahG expression in the dominant eto3 mutant attenuated ethylene-dependent PR4 expression and rescued the O3-induced HR (hypersensitive response) cell death phenotype exhibited by eto3 plants. Our results suggest that both SA and ethylene act in concert to influence cell death in O3-sensitive genotypes, and that O3-induced ethylene production is dependent on SA.     

Multiple roles of WIN3 in regulating disease resistance, cell death, and flowering time in Arabidopsis

The salicylic acid (SA) regulatory gene HOPW1-1-INTERACTING3 (WIN3) was previously shown to confer resistance to the biotrophic pathogen Pseudomonas syringae. Here, we report that WIN3 controls broad-spectrum disease resistance to the necrotrophic pathogen Botrytis cinerea and contributes to basal defense induced by flg22, a 22-amino acid peptide derived from the conserved region of bacterial flagellin proteins. Genetic analysis indicates that WIN3 acts additively with several known SA regulators, including PHYTOALEXIN DEFICIENT4, NONEXPRESSOR OF PR GENES1 (NPR1), and SA INDUCTION-DEFICIENT2, in regulating SA accumulation, cell death, and/or disease resistance in the Arabidopsis (Arabidopsis thaliana) mutant acd6-1. Interestingly, expression of WIN3 is also dependent on these SA regulators and can be activated by cell death, suggesting that WIN3-mediated signaling is interconnected with those derived from other SA regulators and cell death. Surprisingly, we found that WIN3 and NPR1 synergistically affect flowering time via influencing the expression of flowering regulatory genes FLOWERING LOCUS C and FLOWERING LOCUS T. Taken together, our data reveal that WIN3 represents a novel node in the SA signaling networks to regulate plant defense and flowering time. They also highlight that plant innate immunity and development are closely connected processes, precise regulation of which should be important for the fitness of plants.     

The pepper mannose-binding lectin gene CaMBL1 is required to regulate cell death and defense responses to microbial pathogens

Plant mannose-binding lectins (MBLs) are crucial for plant defense signaling during pathogen attack by recognizing specific carbohydrates on pathogen surfaces. In this study, we isolated and functionally characterized a novel pepper (Capsicum annuum) MBL gene, CaMBL1, from pepper leaves infected with Xanthomonas campestris pv vesicatoria (Xcv). The CaMBL1 gene contains a predicted Galanthus nivalis agglutinin-related lectin domain responsible for the recognition of high-mannose N-glycans but lacks a middle S-locus glycoprotein domain and a carboxyl-terminal PAN-Apple domain. The CaMBL1 protein exhibits binding specificity for mannose and is mainly localized to the plasma membrane. Immunoblotting using a CaMBL1-specific antibody revealed that CaMBL1 is strongly expressed and accumulates in pepper leaves during avirulent Xcv infection. The transient expression of CaMBL1 induces the accumulation of salicylic acid (SA), the activation of defense-related genes, and the cell death phenotype in pepper. The G. nivalis agglutinin-related lectin domain of CaMBL1 is responsible for cell death induction. CaMBL1-silenced pepper plants are more susceptible to virulent or avirulent Xcv infection compared with unsilenced control plants, a phenotype that is accompanied by lowered reactive oxygen species accumulation, reduced expression of downstream SA target genes, and a concomitant decrease in SA accumulation. In contrast, CaMBL1 overexpression in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to Pseudomonas syringae pv tomato and Alternaria brassicicola infection. Together, these data suggest that CaMBL1 plays a key role in the regulation of plant cell death and defense responses through the induction of downstream defense-related genes and SA accumulation after the recognition of microbial pathogens.     

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.     

Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.     

Suppression and Restoration of Lesion Formation in Arabidopsis lsd Mutants

Systemic acquired resistance (SAR) is a broad-spectrum, systemic defense response that is activated in many plant species after pathogen infection. We have previously described Arabidopsis mutants that constitutively express SAR and concomitantly develop lesions simulating disease (lsd). Here, we describe two new mutants, lsd6 and lsd7, that develop spontaneous necrotic lesions and possess elevated levels of salicylic acid (SA) as well as heightened disease resistance, similar to the previously characterized lsd and accelerated cell death (acd2) mutants. Genetic analysis of lsd6 and lsd7 showed that the mutant phenotypes segregated as simple dominant traits. When crossed with transgenic Arabidopsis plants containing the SA-degrading enzyme salicylate hydroxylase, the F1 progeny showed suppression of both SAR gene expression and resistance. In addition, salicylate hydroxylase suppressed lesion formation in the F1 progeny, suggesting that SA or some SA-dependent process may have a role in pathogen-associated cell death. Surprisingly, lesions were restored in the lsd6 F1 progeny after the application of either 2,6-dichloroisonicotinic acid or SA. Lesions were not restored by treatment with either compound in the lsd7 F1 plants. Our findings demonstrate that steps early in the signal transduction pathway leading to SAR and disease resistance are potentiated by later events, suggesting feedback control of lesion formation.     

Yeast Cell Wall Extract Induces Disease Resistance against Bacterial and Fungal Pathogens in Arabidopsis thaliana and Brassica Crop

Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.     

ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis

The molecular interactions between Arabidopsis and the pathogenic powdery mildew Golovinomyces cichoracearum were studied by characterizing a disease-resistant Arabidopsis mutant atg2-2. The atg2-2 mutant showed enhanced resistance to powdery mildew and dramatic mildew-induced cell death as well as early senescence phenotypes in the absence of pathogens. Defense-related genes were constitutively activated in atg2-2. In atg2-2 mutants, spontaneous cell death, early senescence and disease resistance required the salicylic acid (SA) pathway, but interestingly, mildew-induced cell death was not fully suppressed by inactivation of SA signaling. Thus, cell death could be uncoupled from disease resistance, suggesting that cell death is not sufficient for resistance to powdery mildew. ATG2 encodes autophagy-related 2, a protein known to be involved in the early steps of autophagosome biogenesis. The atg2-2 mutant exhibited typical autophagy defects in autophagosome formation. Furthermore, mutations in several other ATG genes, including ATG5, ATG7 and ATG10, exhibited similar powdery mildew resistance and mildew-induced cell death phenotypes. Taken together, our findings provide insights into the role of autophagy in cell death and disease resistance, and may indicate general links between autophagy, senescence, programmed cell death and defense responses in plants.