Implications of the mesophyll conductance to CO2 for photosynthesis and water-use efficiency during long-term water stress and recovery in two contrasting Eucalyptu species

Water stress (WS) slows growth and photosynthesis (An), but most knowledge comes from short-time studies that do not account for longer term acclimation processes that are especially relevant in tree species. Using two Eucalyptus species that contrast in drought tolerance, we induced moderate and severe water deficits by withholding water until stomatal conductance (gsw) decreased to two pre-defined values for 24 d, WS was maintained at the target gsw for 29 d and then plants were re-watered. Additionally, we developed new equations to simulate the effect on mesophyll conductance (gm) of accounting for the resistance to refixation of CO2. The diffusive limitations to CO2, dominated by the stomata, were the most important constraints to An. Full recovery of An was reached after re-watering, characterized by quick recovery of gm and even higher biochemical capacity, in contrast to the slower recovery of gsw. The acclimation to long-term WS led to decreased mesophyll and biochemical limitations, in contrast to studies in which stress was imposed more rapidly. Finally, we provide evidence that higher gm under WS contributes to higher intrinsic water-use efficiency (iWUE) and reduces the leaf oxidative stress, highlighting the importance of gm as a target for breeding/genetic engineering.

New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Functional response of Ulmus minor Mill. to drought, flooding and dutch elm disease

Ulmus minor es una especie arbórea originaria de Europa cuyas poblaciones han sido diezmadas por el hongo patógeno causante de la enfermedad de la grafiosis. La conservación de los olmos exige plantearse su propagación a través de plantaciones y conocer mejor su ecología y biología. Ulmus minor es un árbol de ribera, pero frecuentemente se encuentra alejado del cauce de arroyos y ríos, donde la capa freática sufre fuertes oscilaciones. Por ello, nuestra hipótesis general es que esta especie es moderadamente resistente tanto a la inundación como a la sequía. El principal objetivo de esta tesis doctoral es entender desde un punto de vista funcional la respuesta de U. minor a la inundación, la sequía y la infección por O. novo-ulmi; los factores que posiblemente más influyen en la distribución actual de U. minor. Con este objetivo se persigue dar continuidad a los esfuerzos de conservación de esta especie que desde hace años se dedican en varios centros de investigación a nivel mundial, ya que, entender mejor los mecanismos que contribuyen a la resistencia de U. minor ante la inoculación con O. novo-ulmi y factores de estrés abiótico ayudará en la selección y propagación de genotipos resistentes a la grafiosis. Se han planteado tres experimentos en este sentido. Primero, se ha comparado la tolerancia de brinzales de U. minor y U. laevis – otro olmo ibérico – a una inmersión controlada con el fin de evaluar su tolerancia a la inundación y comprender los mecanismos de aclimatación. Segundo, se ha comparado la tolerancia de brinzales de U. minor y Quercus ilex – una especie típica de ambientes Mediterránea secos – a la falta de agua en el suelo con el fin de evaluar el grado de tolerancia y los mecanismos de aclimatación a la sequía. El hecho de comparar dos especies contrastadas responde al interés en entender mejor cuales son los procesos que conducen a la muerte de una planta en condiciones de sequía – asunto sobre el que hay una interesante discusión desde hace algunos años. En tercer lugar, con el fin de entender mejor la resistencia de algunos genotipos de U. minor a la grafiosis, se han estudiado las diferencias fisiológicas y químicas constitutivas e inducidas por O. novo-ulmi entre clones de U. minor seleccionados a priori por su variable grado de resistencia a esta enfermedad. En el primer experimento se observó que los brinzales de U. minor sobrevivieron 60 días inmersos en una piscina con agua no estancada hasta una altura de 2-3 cm por encima del cuello de la raíz. A los 60 días, los brinzales de U. laevis se sacaron de la piscina y, a lo largo de las siguientes semanas, fueron capaces de recuperar las funciones fisiológicas que habían sido alteradas anteriormente. La conductividad hidráulica de las raíces y la tasa de asimilación de CO2 neta disminuyeron en ambas especies. Por el contrario, la tasa de respiración de hojas, tallos y raíces aumentó en las primeras semanas de la inundación, posiblemente en relación al aumento de energía necesario para desarrollar mecanismos de aclimatación a la inundación, como la hipertrofia de las lenticelas que se observó en ambas especies. Por ello, el desequilibrio del balance de carbono de la planta podría ser un factor relevante en la mortalidad de las plantas ante inundaciones prolongadas. Las plantas de U. minor (cultivadas en envases de 16 litros a media sombra) sobrevivieron por un prolongado periodo de tiempo en verano sin riego; la mitad de las plantas murieron tras 90 días sin riego. El cierre de los estomas y la pérdida de hojas contribuyeron a ralentizar las pérdidas de agua y tolerar la sequía en U. minor. Las obvias diferencias en tolerancia a la sequía con respecto a Q. ilex se reflejaron en la distinta capacidad para ralentizar la aparición del estrés hídrico tras dejar de regar y para transportar agua en condiciones de elevada tensión en el xilema. Más relevante es que las plantas con evidentes síntomas de decaimiento previo a su muerte exhibieron pérdidas de conductividad hidráulica en las raíces del 80% en ambas especies, mientras que las reservas de carbohidratos apenas variaron y lo hicieron de forma desigual en ambas especies. Árboles de U. minor de 5 y 6 años de edad (plantados en eras con riego mantenido) exhibieron una respuesta a la inoculación con O. novo-ulmi consistente con ensayos previos de resistencia. La conductividad hidráulica del tallo, el potencial hídrico foliar y la tasa de asimilación de CO2 neta disminuyeron significativamente en relación a árboles inoculados con agua, pero solo en los clones susceptibles. Este hecho enlaza con el perfil químico “más defensivo” de los clones resistentes, es decir, con los mayores niveles de suberina, ácidos grasos y compuestos fenólicos en estos clones que en los susceptibles. Ello podría restringir la propagación del hongo en el árbol y preservar el comportamiento fisiológico de los clones resistentes al inocularlos con el patógeno. Los datos indican una respuesta fisiológica común de U. minor a la inundación, la sequía y la infección por O. novo-ulmi: pérdida de conductividad hidráulica, estrés hídrico y pérdida de ganancia neta de carbono. Pese a ello, U. minor desarrolla varios mecanismos que le confieren una capacidad moderada para vivir en suelos temporalmente anegados o secos. Por otro lado, el perfil químico es un factor relevante en la resistencia de ciertos genotipos a la grafiosis. Futuros estudios deberían examinar como este perfil químico y la resistencia a la grafiosis se ven alteradas por el estrés abiótico. ABSTRACT Ulmus minor is a native European elm species whose populations have been decimated by the Dutch elm disease (DED). An active conservation of this species requires large-scale plantations and a better understanding of its biology and ecology. U. minor generally grows close to water channels. However, of the Iberian riparian tree species, U. minor is the one that spread farther away from rivers and streams. For these reasons, we hypothesize that this species is moderately tolerant to both flooding and drought stresses. The main aim of the present PhD thesis is to better understand the functional response of U. minor to the abiotic stresses – flooding and drought – and the biotic stress – DED – that can be most influential on its distribution. The overarching goal is to aid in the conservation of this emblematic species through a better understanding of the mechanisms that contribute to resistance to abiotic and biotic stresses; an information that can help in the selection of resistant genotypes and their expansion in large-scale plantations. To this end, three experiments were set up. First, we compared the tolerance to experimental immersion between seedlings of U. minor and U. laevis – another European riparian elm species – in order to assess their degree of tolerance and understand the mechanisms of acclimation to this stress. Second, we investigated the tolerance to drought of U. minor seedlings in comparison with Quercus ilex (an oak species typical of dry Mediterranean habitats). Besides assessing and understanding U. minor tolerance to drought at the seedling stage, the aim was to shed light into the functional alterations that trigger drought-induced plant mortality – a matter of controversy in the last years. Third, we studied constitutive and induced physiological and biochemical differences among clones of variable DED resistance, before and following inoculation with Ophiostoma novo-ulmi. The goal is to shed light into the factors of DED resistance that is evident in some genotypes of U. minor, but not others. Potted seedlings of U. minor survived for 60 days immersed in a pool with running water to approximately 2-3 cm above the stem collar. By this time, U. minor seedlings died, whereas U. laevis seedlings moved out of the pool were able to recover most physiological functions that had been altered by flooding. For example, root hydraulic conductivity and leaf photosynthetic CO2 uptake decreased in both species; while respiration initially increased with flooding in leaves, stems and roots possibly to respond to energy demands associated to mechanisms of acclimation to soil oxygen deficiency; as example, a remarkable hypertrophy of lenticels was soon observed in flooded seedlings of both species. Therefore, the inability to maintain a positive carbon balance somehow compromises seedling survival under flooding, earlier in U. minor than U. laevis, partly explaining their differential habitats. Potted seedlings of U. minor survived for a remarkable long time without irrigation – half of plants dying only after 90 days of no irrigation in conditions of high vapour pressure deficit typical of summer. Some mechanisms that contributed to tolerate drought were leaf shedding and stomata closure, which reduced water loss and the risk of xylem cavitation. Obviously, U. minor was less tolerant to drought than Q. ilex, differences in drought tolerance resulting mostly from the distinct capacity to postpone water stress and conduct water under high xylem tension among species. More relevant was that plants of both species exhibited similar symptoms of root hydraulic failure (i.e. approximately 80% loss of hydraulic conductivity), but a slight and variable depletion of non-structural carbohydrate reserves preceding dieback. Five- and six-year-old trees of U. minor (planted in the field with supplementary watering) belonging to clones of contrasted susceptibility to DED exhibited a different physiological response to inoculation with O. novo-ulmi. Stem hydraulic conductivity, leaf water potential and photosynthetic CO2 uptake decreased significantly relative to control trees inoculated with water only in DED susceptible clones. This is consistent with the “more defensive” chemical profile observed in resistant clones, i.e. with higher levels of saturated hydrocarbons (suberin and fatty acids) and phenolic compounds than in susceptible clones. These compounds could restrict the spread of O. novo-ulmi and contribute to preserving the near-normal physiological function of resistant trees when exposed to the pathogen. These results evidence common physiological responses of U. minor to flooding, drought and pathogen infection leading to xylem water disruption, leaf water stress and reduced net carbon gain. Still, seedlings of U. minor develop various mechanisms of acclimation to abiotic stresses that can play a role in surviving moderate periods of flood and drought. The chemical profile appears to be an important factor for the resistance of some genotypes of U. minor to DED. How abiotic stresses such as flooding and drought affect the capacity of resistant U. minor clones to face O. novo-ulmi is a key question that must be contemplated in future research.

Compromised disease resistance in saponin-deficient plants

Saponins are glycosylated plant secondary metabolites found in many major food crops [Price, K. R., Johnson, I. T. & Fenwick, G. R. (1987) CRC Crit. Rev. Food Sci. Nutr. 26, 27–133]. Because many saponins have potent antifungal properties and are present in healthy plants in high concentrations, these molecules may act as preformed chemical barriers to fungal attack. The isolation of plant mutants defective in saponin biosynthesis represents a powerful strategy for evaluating the importance of these compounds in plant defense. The oat root saponin avenacin A-1 fluoresces under ultraviolet illumination [Crombie, L., Crombie, W. M. L. & Whiting, D. A. (1986) J. Chem. Soc. Perkins 1, 1917–1922], a property that is extremely rare among saponins. Here we have exploited this fluorescence to isolate saponin-deficient (sad) mutants of a diploid oat species, Avena strigosa. These sad mutants are compromised in their resistance to a variety of fungal pathogens, and a number of lines of evidence suggest that this compromised disease resistance is a direct consequence of saponin deficiency. Because saponins are widespread throughout the plant kingdom, this group of secondary metabolites may have general significance as antimicrobial phytoprotectants.

CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus

Cry proteins produced by Bacillus thuringiensis are selective biodegradable insecticides used increasingly in bacterial insecticides and transgenic plants as alternatives to synthetic chemical insecticides. However, the potential for development of resistance and cross-resistance in target insect populations to Cry proteins used alone or in combination threatens the more widespread use of this novel pest control technology. Here we show that high levels of resistance to CryIV proteins in larvae of the mosquito, Culex quinquefasciatus, can be suppressed or reduced markedly by combining these proteins with sublethal quantities of CytA, a cytolytic endotoxin of B. thuringiensis. Resistance at the LC95 level of 127-fold for a combination of three CryIV toxins (CryIVA, B, and D), resulting from 60 generations of continuous selection, was completely suppressed by combining sporulated powders of CytA in a 1:3 ratio with sporulated powders of a CryIVA, CryIVB, and CryIVD strain. Combining the CytA strain with a CryIVA and CryIVB strain also completely suppressed mosquito resistance of 217-fold to the latter toxins at the LC95 level, whereas combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from greater than 1,000-fold to less than 8-fold. The CytA/CryIV model provides a potential molecular genetic strategy for engineering resistance management for Cry proteins directly into bacterial insecticides and transgenic plants.

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

Insecticide resistance resulting from an absence of target-site gene product

Genetic changes in insects that lead to insecticide resistance include point mutations and up-regulation/amplification of detoxification genes. Here, we report a third mechanism, resistance caused by an absence of gene product. Mutations of the Methoprene-tolerant (Met) gene of Drosophila melanogaster result in resistance to both methoprene, a juvenile hormone (JH) agonist insecticide, and JH. Previous results have demonstrated a mechanism of resistance involving an intracellular JH binding protein that has reduced ligand affinity in Met flies. We show that a γ-ray induced allele, Met27, completely lacks Met transcript during the insecticide-sensitive period in development. Although Met27 homozygotes have reduced oogenesis, they are viable, demonstrating that Met is not a vital gene. Most target-site resistance genes encode vital proteins and thus have few mutational changes that permit both resistance and viability. In contrast, resistance genes such as Met that encode nonvital insecticide target proteins can have a variety of mutational changes that result in an absence of functional gene product and thus should show higher rates of resistance evolution.

Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50°C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Alternatively spliced N resistance gene transcripts: Their possible role in tobacco mosaic virus resistance

The N gene, a member of the Toll-IL-1 homology region–nucleotide binding site–leucine-rich repeat region (LRR) class of plant resistance genes, encodes two transcripts, NS and NL, via alternative splicing of the alternative exon present in the intron III. The NS transcript, predicted to encode the full-length N protein containing the Toll-IL-1 homology region, nucleotide binding site, and LRR, is more prevalent before and for 3 hr after tobacco mosaic virus (TMV) infection. The NL transcript, predicted to encode a truncated N protein (Ntr) lacking 13 of the 14 repeats of the LRR, is more prevalent 4–8 hr after TMV infection. Plants harboring a cDNA-NS transgene, capable of encoding an N protein but not an Ntr protein, fail to exhibit complete resistance to TMV. Transgenic plants containing a cDNA-NS-bearing intron III and containing 3′ N-genomic sequences, encoding both NS and NL transcripts, exhibit complete resistance to TMV. These results suggest that both N transcripts and presumably their encoded protein products are necessary to confer complete resistance to TMV.

Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants

Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Independent deletions of a pathogen-resistance gene in Brassica and Arabidopsis

Plant disease resistance (R) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most R loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast, the resistance to Pseudomonas syringae pathovar maculicola (RPM1) bacterial resistance gene is completely absent (rpm1-null) in 5/5 Arabidopsis thaliana accessions that lack RPM1 function. The rpm1-null locus contains a 98-bp segment of unknown origin in place of the RPM1 gene. We undertook comparative mapping of RPM1 and flanking genes in Brassica napus to determine the ancestral state of the RPM1 locus. We cloned two B. napus RPM1 homologs encoding hypothetical proteins with ≈81% amino acid identity to Arabidopsis RPM1. Collinearity of genes flanking RPM1 is conserved between B. napus and Arabidopsis. Surprisingly, we found four additional B. napus loci in which the flanking marker synteny is maintained but RPM1 is absent. These B. napus rpm1-null loci have no detectable nucleotide similarity to the Arabidopsis rpm1-null allele. We conclude that RPM1 evolved before the divergence of the Brassicaceae and has been deleted independently in the Brassica and Arabidopsis lineages. These results suggest that functional polymorphism at R gene loci can arise from gene deletions.

The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response

Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (avr) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (R) gene products. Members of the NBS-LRR class of R genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBS-LRR protein from Arabidopsis thaliana that confers resistance to Pseudomonas syringae expressing either avrRpm1 or avrB. RPM1 protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

Enhanced resistance in STAT6-deficient mice to infection with ectromelia virus

We inoculated BALB/c mice deficient in STAT6 (STAT6−/−) and their wild-type (wt) littermates (STAT6+/+) with the natural mouse pathogen, ectromelia virus (EV). STAT6−/− mice exhibited increased resistance to generalized infection with EV when compared with STAT6+/+ mice. In the spleens and lymph nodes of STAT6−/− mice, T helper 1 (Th1) cytokines were induced at earlier time points and at higher levels postinfection when compared with those in STAT6+/+ mice. Elevated levels of NO were evident in plasma and splenocyte cultures of EV-infected STAT6−/− mice in comparison with STAT6+/+ mice. The induction of high levels of Th1 cytokines in the mutant mice correlated with a strong natural killer cell response. We demonstrate in genetically susceptible BALB/c mice that the STAT6 locus is critical for progression of EV infection. Furthermore, in the absence of this transcription factor, the immune system defaults toward a protective Th1-like response, conferring pronounced resistance to EV infection and disease progression.

A Novel Gene, pmgA, Specifically Regulates Photosystem Stoichiometry in the Cyanobacterium Synechocystis Species PCC 6803 in Response to High Light1

Previously, we identified a novel gene, pmgA, as an essential factor to support photomixotrophic growth of Synechocystis species PCC 6803 and reported that a strain in which pmgA was deleted grew better than the wild type under photoautotrophic conditions. To gain insight into the role of pmgA, we investigated the mutant phenotype of pmgA in detail. When low-light-grown (20 μE m−2 s−1) cells were transferred to high light (HL [200μE m−2 s−1]), pmgA mutants failed to respond in the manner typically associated with Synechocystis. Specifically, mutants lost their ability to suppress accumulation of chlorophyll and photosystem I and, consequently, could not modulate photosystem stoichiometry. These phenotypes seem to result in enhanced rates of photosynthesis and growth during short-term exposure to HL. Moreover, mixed-culture experiments clearly demonstrated that loss of pmgA function was selected against during longer-term exposure to HL, suggesting that pmgA is involved in acquisition of resistance to HL stress. Finally, early induction of pmgA expression detected by reverse transcriptase-PCR upon the shift to HL led us to conclude that pmgA is the first gene identified, to our knowledge, as a specific regulatory factor for HL acclimation.