Background: We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes.

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries – even millennia – ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.

Class I hydrophobins are a unique family of fungal proteins that form a polymeric, water-repellent monolayer on the surface of structures such as spores and fruiting bodies. Similar monolayers are being discovered on an increasing range of important microorganisms. Hydrophobin monolayers are amphipathic and particularly robust, and they reverse the wettability of the surface on which they are formed. There are also significant similarities between these polymers and amyloid-like fibrils. However, structural information on these proteins and the rodlets they form has been elusive. Here, we describe the three-dimensional structure of the monomeric form of the class I hydrophobin EAS. EAS forms a ␤-barrel structure punctuated by several disordered regions and displays a complete segregation of charged and hydrophobic residues on its surface. This structure is consistent with its ability to form an amphipathic polymer. By using this structure, together with data from mutagenesis and previous biophysical studies, we have been able to propose a model for the polymeric rodlet structure adopted by these proteins. X-ray fiber diffraction data from EAS rodlets are consistent with our model. Our data provide molecular insight into the nature of hydrophobin rodlet films and extend our understanding of the fibrillar ␤-structures that continue to be discovered in the protein world. amyloid ͉ NMR ͉ polymer H ydrophobins are a large family of secreted, low-molecularmass (7-9 kDa) proteins unique to filamentous fungi. There is little amino acid sequence similarity between hydrophobins, except for a characteristic pattern of eight cysteine residues that form four intramolecular disulfide bonds (1, 2). These proteins have remarkable biophysical properties and function by selfassembling into amphipathic polymeric films at the interface between hydrophobic and hydrophilic surfaces (3). The surfactive and amphipathic properties of hydrophobins facilitate the formation of essential aerial structures such as hyphae, spores, and fruiting bodies (4).Two classes of hydrophobins have been identified based on their hydrophobicity plots and physical properties (5). For class I hydrophobins, the polymer film comprises cylindrical rodlets with dimensions of Ϸ10 ϫ 100-250 nm and their outward-facing hydrophobic surface has extremely low wettability (6, 7). These films are very robust; they are resistant to boiling in detergents or strong alkalis (8, 9). The morphology of isolated rodlets is reminiscent of amyloid fibrils isolated from diseased tissue and formed in vitro. Reconstituted rodlets stained with Congo red give the green-gold birefringence characteristic of similarly stained amyloid fibers and circular dichroism (CD) data indicate that the rodlets contain extensive ␤-structure, suggesting that rodlets and amyloid fibrils have structural features in common (10, 11, **). Class II hydrophobin films are significantly less robust and lack the rodlet morphology of class I hydrophobins (12, 13).Class I hydrophobins, once solubilized, will spo...

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