Secreted surface proteins are an innate immune defense component employed by animals to inhibit invading microbes. Surface proteins have not been documented in plants, even though the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum, and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete pathogen Peronospora tabacina. We investigated the site of phylloplanin biosynthesis using biochemical techniques. These techniques included radiolabeling of detached trichome glands, radiolabeling of epidermal peels, analysis of leaf water washes of various Nicotiana plants, and examination of guttation fluid, leaf vein contents, and extracellular fluid. From these experiments, we tentatively conclude that phylloplanins are produced by hydathodes, or an unknown surface secreting system, but not by glandular secreting trichomes. Future experiments with the phylloplanin promoter, whose elucidation is described herein, and its fusion to a reporter gene (GUS or GFP), will undoubtedly provide further insight into the location of phylloplanin biosynthesis and deposition. We suggest that the hydrophobic nature of phylloplanins aids in their dispersal over the leaf surface. Phylloplanins constitute a first-point-of-contact, rapid response, innate immune deterrent to pathogen establishment on N. tabacum leaf surfaces, and are the first studied representatives of a novel protein class in the plant kingdom. Further study of leaf surface proteins is justified to understand further their roles in plant defense, and to investigate their potential in agricultural biotechnology. Additionally, we describe miscellaneous observations we have made during the course of this research. Low molecular mass proteins (as yet uncharacterized) are washed from leaf surfaces of sunflower, soybean, and other plants. Pathogenesis-related (PR-)-5a, a known antifungal protein, was found to be present on the leaf surfaces of healthy plants, although its function there remains unknown. A phylloplanin homologue from Arabidopsis appears to be antibacterial. Further study of this protein is warranted. We note that proteins can also be recovered from N. tabacum root surfaces, or the rhizoplane, but we have not further characterized these proteins. In summary, novel surface-accumulated proteins, termed phylloplanins, and the gene encoding these have been discovered in N. tabacum. An antifungal function for phylloplanins is reported, and evidence was found for a unique mechanism of surface deposition.

Made available in DSpace on 2015-03-26T13:37:42Z (GMT). No. of bitstreams: 1 texto completo.pdf: 611413 bytes, checksum: d8119b46b042a6c0388e233d2daa1dcc (MD5) Previous issue date: 2010-02-23 / Looking for promising microorganisms for the control of coffee leaf rust, caused by Hemileia vastatrix, were isolated phylloplane residents from coffee leaves healthy by simply washing the sheet or by using ultrasound, with subsequent dilution. Were also selected bacteria capable of forming endospores, yielding a total of 217 isolates. After isolation were carried out four tests for selection of these organisms as biocontrol agents of rust. The first test conducted was the germination of urediniospores of H. vastatrix on glass slide, comparing the germination in distilled water and germination when a drop of each of the isolates obtained was deposited, from this experiment we selected 33 isolates with an inhibitory effect on germination. Subsequently, isolates were tested on leaf discs, as its capacity to suppress disease progression. The 33 isolates were sprayed on leaves of coffee plants at three different times in relation to inoculation with H. vastatrix (24 hours before, simultaneously and 24 hours later). Four isolates were considered capable of reducing the severity of the disease in the conditions of the experiment. These four isolates were tested on detached leaves to confirm the results, again observing reductions in disease severity compared to control, which contained only distilled water. The last experiment was conducted in green-house, comparing the four isolates with the controls (sprayed only with distilled water and with the systemic fungicide epoxiconazol). The four isolates provided the level of disease control similar to that obtained by the use of fungicide (approximately 100%). The identification based on fatty acid profile of the isolates identified UFV 025, UFV UFV 033 and 080 as Bacillus cereus, isolate UFV and 070 as Bacillus sphaericus. With the results of the experiments we can conclude that resident bacteria in the phylloplane of coffee can be isolated and selected as potential biocontrol agents of rust. / Procurando-se microorganismos promissores para o controle da ferrugem do cafeeiro, causada por Hemileia vastatrix, foram isolados residentes do filoplano de folhas de café sadias por simples lavagem de folha ou por utilização de ultra-som, com posterior diluição. Também foram selecionadas bactérias capazes de formar endósporos, obtendo-se um total de 217 isolados. Após o isolamento foram realizados quatro ensaios para seleção destes organismos como agentes de biocontrole da ferrugem. O primeiro ensaio realizado foi de germinação de urediniósporos de H. vastatrix em lâmina de vidro, comparando-se a germinação em água destilada e a germinação quando foi depositada uma gota de cada um dos isolados obtidos, deste experimento foram selecionados 33 isolados com efeito inibitório na germinação. Posteriormente os isolados foram testados, em discos de folhas, quanto a sua capacidade em suprimir o avanço da doença. Os 33 isolados foram atomizados em folhas de cafeeiro em três diferentes tempos em relação à inoculação de H. vastatrix (24 horas antes, simultaneamente e 24 horas depois). Quatro isolados foram selecionados e considerados capazes de reduzir a severidade da doença nas condições do experimento. Esses quatro isolados foram testados em folhas destacadas para confirmação dos resultados, novamente observando-se redução na intensidade da doença em relação à testemunha, que continha apenas água destilada. O último experimento foi conduzido em casa-de-vegetação, comparando-se os quatro isolados com as testemunhas (pulverização apenas com água destilada e com o fungicida sistêmico epoxiconazol). Os quatro isolados proporcionaram o nível de controle da doença similar ao obtido pelo uso do fungicida (aproximadamente 100%). A identificação baseada no perfil de ácidos graxos identificou os isolados UFV 025, UFV 033 e UFV 080 como Bacillus cereus, e o isolado UFV 070 como Bacillus sphaericus. Com os resultados dos experimentos podemos concluir que bactérias residentes do filoplano de cafeeiro podem ser isoladas e selecionadas como potenciais agentes de biocontrole de ferrugem