Dominique Roby

Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?

The identification of several lesion mimic mutants (LMM) that misregulate cell death constitutes a powerful tool to unravel programmed cell death (PCD) pathways in plants, particularly the hypersensitive response (HR), a form of PCD associated with resistance to pathogens. Recently, the characterization of novel LMM has enabled genes that might regulate cell death programmes to be identified as well as the dissection of defense signaling pathways and of crosstalk between multiple pathways in ways that might not be possible by studying the responses of wild-type plants to pathogens.

The Transcription Factors WRKY11 and WRKY17 Act as Negative Regulators of Basal Resistance in Arabidopsis thaliana

Transcription factors are believed to play a pivotal role in the activation and fine-tuning of plant defense responses, but little is known about the exact function of individual transcription factors in this process. We analyzed the role of the IId subfamily of WRKY transcription factors in the regulation of basal resistance to Pseudomonas syringae pv tomato (Pst). The expression of four members of the subfamily was induced upon challenge with virulent and avirulent strains of Pst. Mutant analyses revealed that loss of WRKY11 function increased resistance toward avirulent and virulent Pst strains and that resistance was further enhanced in wrky11 wrky17 double mutant plants. Thus, WRKY11 and WRKY17 act as negative regulators of basal resistance to Pst. Genome-wide expression analysis and expression studies of selected genes in single and double mutants demonstrated that both transcription factors modulate transcriptional changes in response to pathogen challenge. Depending on the target gene, WRKY11 and WRKY17 act either specifically or in a partially redundant manner. We demonstrate complex cross-regulation within the IId WRKY subfamily and provide evidence that both WRKY transcription factors are involved in the regulation of Pst-induced jasmonic acid–dependent responses. These results provide genetic evidence for the importance of WRKY11 and WRKY17 in plant defense.

HLM1, an Essential Signaling Component in the Hypersensitive Response, Is a Member of the Cyclic Nucleotide–Gated Channel Ion Channel Family

The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide–gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K+ and Na+ and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.

A MYB transcription factor regulates very-long-chain fatty acid biosynthesis for activation of the hypersensitive cell death response in Arabidopsis.

Plant immune responses to pathogen attack include the hypersensitive response (HR), a form of programmed cell death occurring at invasion sites. We previously reported on Arabidopsis thaliana MYB30, a transcription factor that acts as a positive regulator of a cell death pathway conditioning the HR. Here, we show by microarray analyses of Arabidopsis plants misexpressing MYB30 that the genes encoding the four enzymes forming the acyl-coA elongase complex are putative MYB30 targets. The acyl-coA elongase complex synthesizes very-long-chain fatty acids (VLCFAs), and the accumulation of extracellular VLCFA-derived metabolites (leaf epidermal wax components) was affected in MYB30 knockout mutant and overexpressing lines. In the same lines, a lipid extraction procedure allowing high recovery of sphingolipids revealed changes in VLCFA contents that were amplified in response to inoculation. Finally, the exacerbated HR phenotype of MYB30-overexpressing lines was altered by the loss of function of the acyl-ACP thioesterase FATB, which causes severe defects in the supply of fatty acids for VLCFA biosynthesis. Based on these findings, we propose a model in which MYB30 modulates HR via VLCFAs by themselves, or VLCFA derivatives, as cell death messengers in plants.

A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack

Hypersensitive response (HR) is a programmed cell death that is commonly associated with disease resistance in plants. Among the different HR-related early induced genes, the AtMYB30 gene is specifically, rapidly, and transiently expressed during incompatible interactions between Arabidopsis and bacterial pathogens. Its expression was also shown to be deregulated in Arabidopsis mutants affected in the control of cell death initiation. Here, we demonstrate that overexpression in Arabidopsis and tobacco of AtMYB30 (i) accelerates and intensifies the appearance of the HR in response to different avirulent bacterial pathogens, (ii) causes HR-like responses to virulent strains, and (iii) increases resistance against different bacterial pathogens, and a virulent biotrophic fungal pathogen, Cercospora nicotianae. In antisense AtMYB30 Arabidopsis lines, HR cell death is strongly decreased or suppressed in response to avirulent bacterial strains, resistance against different bacterial pathogens decreased, and the expression of HR- and defense-related genes was altered. Taken together, these results strongly suggest that AtMYB30 is a positive regulator of hypersensitive cell death.

Markers for hypersensitive response and senescence show distinct patterns of expression.

Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.

Pathogen-Induced Elicitin Production in Transgenic Tobacco Generates a Hypersensitive Response and Nonspecific Disease Resistance

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea–induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.

Activation of hsr203, a Plant Gene Expressed During Incompatible Plant-Pathogen Interactions, Is Correlated with Programmed Cell Death

hsr203J is a tobacco gene whose activation is rapid, highly localized, and specific for incompatible interactions between tobacco and the bacterial pathogen Ralstonia solanacearum. The effect of other hypersensitive response (HR)-inducing pathogens and elicitors has been tested with transgenic plants containing the hsr203J promoter-GUS reporter gene fusion, and confirms the generality of the preferential inducibility of the hsr203J gene promoter during incompatible interactions: bacterial and viral pathogens inducing an HR in tobacco were able to induce the promoter fusion, as were inducers of HR-like responses such as harpin, elicitins, and PopA1 proteins. A tomato hsr203 homologous cDNA was isolated (Lehsr203) and used to examine the effect of avr gene products on the expression of such genes. Lehsr203 was shown to be rapidly and transiently induced in leaves of the tomato Cf-9 line, following Avr9 product infiltration, but not in those of the Cf-0 line. Among potential effectors of HR or resistance such as H2O2, salicylic acid, methyl jasmonate, and 2,6-dichloro-isonicotinic acid (INA), none is able to induce a significant increase in promoter activation. In contrast, heavy metals that cause leaf necrosis can trigger such an activation. In addition, hsr203-GUS fusion expression is detected in transgenic tobacco lines expressing the bO gene and exhibiting spontaneous HR-like lesions. Taken together, these results demonstrate a strong correlation between hsr203 and genetically controlled cell death in tobacco and tomato. The expression of this gene should be a useful marker for programmed cell death occurring in response not only to diverse pathogens, but also to diverse death-triggering extracellular agents.

Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis

The deposition of lignin during plant-pathogen interactions is thought to play a role in plant defence. However, the function of lignification genes in plant disease resistance is poorly understood. In this article, we provide genetic evidence that the primary genes involved in lignin biosynthesis in Arabidopsis, CAD-C and CAD-D, act as essential components of defence to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato, possibly through the salicylic acid defence pathway. Thus, in contrast with cellulose synthesis, whose alteration leads to an increase in disease resistance, alteration of the cell wall lignin content leads directly or indirectly to defects in some defence components.

VASCULAR ASSOCIATED DEATH1, a Novel GRAM Domain–Containing Protein, Is a Regulator of Cell Death and Defense Responses in Vascular Tissues

The hypersensitive response (HR) is a programmed cell death that is commonly associated with plant disease resistance. A novel lesion mimic mutant, vad1 (for vascular associated death1), that exhibits light conditional appearance of propagative HR-like lesions along the vascular system was identified. Lesion formation is associated with expression of defense genes, production of high levels of salicylic acid (SA), and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato. Analyses of the progeny from crosses between vad1 plants and either nahG transgenic plants, sid1, nonexpressor of PR1 (npr1), enhanced disease susceptibility1 (eds1), or non-race specific disease resistance1 (ndr1) mutants, revealed the vad1 cell death phenotype to be dependent on SA biosynthesis but NPR1 independent; in addition, both EDS1 and NDR1 are necessary for the proper timing and amplification of cell death as well as for increased resistance to Pseudomonas strains. VAD1 encodes a novel putative membrane-associated protein containing a GRAM domain, a lipid or protein binding signaling domain, and is expressed in response to pathogen infection at the vicinity of the hypersensitive lesions. VAD1 might thus represent a new potential function in cell death control associated with cells in the vicinity of vascular bundles.