Too hot to defend: a tale of salicylic acid.

Extreme temperatures threaten plant immunity by suppressing the salicylic acid (SA) biosynthesis via unknown mechanisms. Kim et al. demonstrated that suppression of the SA pathway and plant immunity can be rescued by optimised expression of two master immune regulator(s), advancing our prospects for better protecting plants in a warming climate.

Zinc mapping and density imaging of rabbit pancreas endocrine tissue sections using nuclear microscopy.

Nuclear microscopy is a suite of techniques based on a focused beam of MeV protons. These techniques have the unique ability to image density and structural variations in relatively thick tissue sections, map trace elements at the cellular level to the microgram per gram (dry weight) level, and extract quantitative information on these elements. The trace elemental studies can be carried out on unstained freeze-dried tissue sections, thereby minimizing any problems of contamination or redistribution of elements during conventional staining and fixing procedures. The pancreas is a gland with different specialized cells and a complex hormonal activity where trace elements play an important role. For example, zinc has an active role in insulin production, and calcium ions participate in the stimulation and secretion process of insulin. Using nuclear microscopy with a spatial resolution of 1 mum, we have located, using zinc mapping, the islets of Langerhans in freeze-dried normal rabbit tissue sections. The islets of Langerhans contain beta-cells responsible for insulin production. Subsequent quantitative analyses have indicated elevations in most elements within the islets of Langerhans, and significantly so for the concentrations of Zn [3,300 compared to 90 microg/g (dry weight)] and Ca [1,100 compared to 390 microg/g (dry weight)].

RNA-mediated gene silencing of superoxide dismutase (bcsod1) in Botrytis cinerea.

Gene silencing is a powerful tool utilized for identification of gene function and analysis in plants, animals, and fungi. Here, we report the silencing of superoxide dismutase (bcsod1) in Botrytis cinerea through sense and antisense-mediated silencing mechanisms. Because superoxide dismutase (SOD) is a virulence factor, transformants were tested for phenotypic silencing in vitro and reduction in pathogenicity in planta. Plate-based assays with and without paraquat were performed to screen initial silencing efficiency, and a subset of transformants was used for in planta studies of virulence. Transformants exhibiting strongly decreased transcripts levels were recovered with both constructs but none of those exhibited a reduction in virulence in planta. Our investigations may help optimize a high-throughput gene silencing system useful for identifying potential gene targets for future fungal control.

Brightness measurement of an electron impact gas ion source for proton beam writing applications.

We are developing a high brightness nano-aperture electron impact gas ion source, which can create ion beams from a miniature ionization chamber with relatively small virtual source sizes, typically around 100 nm. A prototype source of this kind was designed and successively micro-fabricated using integrated circuit technology. Experiments to measure source brightness were performed inside a field emission scanning electron microscope. The total output current was measured to be between 200 and 300 pA. The highest estimated reduced brightness was found to be comparable to the injecting focused electron beam reduced brightness. This translates into an ion reduced brightness that is significantly better than that of conventional radio frequency ion sources, currently used in single-ended MeV accelerators.

A virus-inducible tobacco gene encoding a glycine-rich protein shares putative regulatory elements with the ribulose bisphosphate carboxylase small subunit gene.

cDNA to an mRNA that is strongly induced in Samsun NN tobacco after tobacco mosaic virus (TMV) infection or salicylic acid treatment was used to probe a genomic blot and to screen a genomic library. The mRNA corresponds to a family of approximately eight genes, four of which were cloned. The sequence of the genes and flanking DNA in two clones was determined. One gene was found to contain an intron of 555 bp; S1-nuclease mapping studies indicated that this gene is expressed. The other gene is interrupted by an intron of 1,954 bp and is probably not expressed after TMV infection. The genes encode a protein of 109 amino acids with a putative N-terminal signal peptide of 26 amino acids. The protein contains a high proportion of glycine (25%) and charged amino acids (29%), suggesting that it may be a cell wall component. A comparison of the upstream sequences of the genes encoding the glycine-rich protein and the pathogenesis-related protein 1a showed only limited homology, although both genes are TMV- and salicylic acid-inducible. However, the upstream sequence of the glycine-rich protein gene contains a 64-bp inverted repeat that occurs in a similar position in the tobacco ribulose bisphosphate carboxylase small subunit gene.

The endopolygalacturonase gene Bcpg1 is required for full virulence of Botrytis cinerea.

Botrytis cinerea, a fungus that causes diseases in over 200 plant species, secretes a number of endopolygalacturonases that have been suggested to be involved in pathogenesis. However, so far the corresponding genes have not been isolated from this fungus. We cloned Bcpg1, encoding endopolygalacturonase, with the pgaII gene from Aspergillus niger as a heterologous probe. The Bcpg1 gene is expressed to similar levels in liquid cultures of B. cinerea containing either 1% polygalacturonic acid or 1% sucrose, and is expressed during infection of tomato leaves. The Bcpg1 gene was eliminated by partial gene replacement, and the resulting mutants were tested for virulence on tomato leaves and fruits, as well as on apple fruits. Although the mutants were still pathogenic and displayed similar primary infections when compared with control strains, a significant decrease in secondary infection, i.e., growth of the lesion beyond the inoculation spot, was observed on all three host tissues. These results indicate that the Bcpg1 gene is required for full virulence.

Cloning and expression of the cutinase A gene of Botrytis cinerea.

Cutinase of Botrytis cinerea has been suggested to play an important role in penetration of host tissues. A protein fraction with cutin hydrolyzing activity was purified from culture filtrates of B. cinerea induced for cutinase activity. An 18-kDa protein in this fraction was identified as cutinase and the corresponding gene cutA was cloned. The gene is present in a single copy in the genome of B. cinerea strain SAS56 and its predicted amino acid sequence shows significant homology (31 to 35% identity) to other fungal cutinases. RNA blot analysis showed that cutA mRNA is induced in vitro by the cutin monomer 16-hydroxyhexadecanoic acid and repressed by glucose. The expression of cutA during infection of tomato leaves is low during early phases of infection, but high when the fungus has colonized the leaf and starts to sporulate.

Cloning and characterization of cDNA of avirulence gene avr9 of the fungal pathogen Cladosporium fulvum, causal agent of tomato leaf mold.

A race-specific peptide elicitor from Cladosporium fulvum induces a hypersensitive response on Cf9 tomato genotypes. We have hypothesized that the avirulence of fungal races on Cf9 genotypes is due to the production of this elicitor by an avirulence gene, avr9. To obtain cDNA clones of the avr9 gene, oligonucleotide probes were designed based on the amino acid sequence determined previously. In northern blot analysis, one oligonucleotide detected an mRNA of 600 nucleotides in tomato-C. fulvum interactions involving fungal races producing the elicitor. A primer extension experiment indicated that the probe hybridized to a region near position 270 of the mRNA. The probe was used to screen a cDNA library made from poly(A)+ RNA from an appropriate compatible tomato-C. fulvum interaction. One clone was obtained corresponding to the mRNA detected by the oligonucleotide probe. Sequence analysis revealed that this clone encoded the avr9 elicitor. By isolating longer clones and by RNA sequencing, the primary structure of the mRNA was determined. The mRNA contains an open reading frame of 63 amino acids, including the sequence of the elicitor at the carboxyterminus. A time course experiment showed that the avr9 mRNA accumulates in a compatible tomato-C. fulvum interaction in correlation with the increase of fungal biomass. The avr9 gene is a single-copy gene that is absent in fungal races which are virulent on tomato Cf9 genotypes. Possible functions of the avirulence gene are discussed.

Cutinase A of Botrytis cinerea is expressed, but not essential, during penetration of gerbera and tomato.

The plant pathogen Botrytis cinerea can infect undamaged plant tissue directly by penetration of the cuticle. This penetration has been suggested to be enzyme-mediated, and an important role for cutinase in the infection process has been proposed. In this study the expression of the cutinase encoding gene cutA of B. cinerea was analyzed using a cutA promoter-GUS reporter gene fusion. Transformants containing the fusion construct were examined for GUS expression on gerbera flowers and tomato fruits. High GUS activity was detected from the onset of conidial germination and during penetration into epidermal cells, indicating that cutA is expressed during the early stages of infection. To determine the biological relevance of cutinase A for successful penetration, cutinase A-deficient mutants were constructed by gene disruption. Pathogenicity of two transformants lacking a functional cutA gene was studied on gerbera flowers and tomato fruits. Their ability to penetrate and cause symptoms was unaltered compared to the wild-type strain. These results exclude an important role for cutinase A during direct penetration of host tissue by B. cinerea.

Tobacco and tomato PR proteins homologous to win and pro-hevein lack the "hevein" domain.

Clones corresponding to tobacco pathogenesis-related (PR) proteins PR-4 and tomato PR protein P2 were isolated from phage cDNA libraries of tobacco infected with tobacco mosaic virus and tomato infected with Cladosporium fulvum, respectively. The probe used in these screenings was a polymerase chain reaction product, synthesized on phage DNA from the tobacco cDNA library, using a synthetic oligonucleotide primer whose sequence corresponded to the partial amino acid sequence available for P2. The different cDNA sequences from the tobacco and tomato clones contained open reading frames for small proteins with 80-90% amino acid sequence identity. Both tobacco PR-4 and tomato P2 are synthesized as precursor proteins, with an N-terminal signal peptide involved in extracellular targeting. The proteins are highly similar to putative wound-induced proteins of potato (win) and to the precursor protein of hevein. However, in contrast to the hevein pro-protein and win proteins, PR-4 and P2 do not contain N-terminal, chitin-binding "hevein" domains. The tobacco and tomato genomes contain a limited number of genes corresponding to PR-4 or P2, whose expression is induced upon infection with the above-mentioned pathogens.