Bacterial Attachment to Leaves Plants and Crops Science
INTRODUCTION
Attachment of bacterial cells to colonizable surfaces is a common event that in many environments absolutely determines the fate of the bacterium. In other environments and for other bacteria, the requirement for attachment is not absolute, but the adherence may still give a selective advantage, and therefore capacity to adhere is a fitness factor. In this context, the term ‘‘surface’’ should be understood broadly, to include not only colonizable tissues or inanimate surfaces but also the surface of other microbes and bacterial cells in, for example, biofilms.
In the case of plant leaf–associated bacteria, both plant pathogenic and saprophytic bacteria harbor a capacity for more or less specific adherence, and they often do carry the genes for expressing attachment-associated surface structures such as pili or fimbriae. But apparently, leaf surface attachment is in most, if not all, cases not an absolutely required trait—at best merely a fitness factor improving the chances for successful colonization in competition with other microbes. This may, however, be decisive in natural environmental conditions where any contribution to fitness determines the outcome of the bacterial competition of the limited resources of the leaf surface or the limited access to the inner leaf tissue.
BACTERIAL STRUCTURES INVOLVED IN ATTACHMENT
When spreading through rain splash, etc., the initial interaction of a bacterial cell with a plant leaf surface is a random event. The distribution of bacteria deposited on a leaf surface after the droplet has dried is, however, far from random. The distribution—high relative bacterial cell densities on trichomes, leaf veins, and stomates—is likely to reflect nonspecific hydrophobic interaction in combination with the degree of access to leaf surface structures, rather than specific interaction of a bacterial adhesin with a receptor, although this type of specificity has not been ruled out.
At any rate, the presence or absence of bacterial appendages, such as pili, fimbriae, flagella, and other bacterial cell surface structures, does influence the ability to attach. Pili and Fimbriae The main, but not exclusive, focus will be on pili and fimbriae produced by various plant-associated, and in most cases plant-pathogenic, bacteria. In contrast to polysaccharides and flagella, most pili or fimbriae have one primary function, and that is attachment.
The direct effect of the presence of these surface appendages is the adhesion of the carrier bacterial cell to surfaces, solid particles, etc. The secondary events following attachment may be very diverse, including successful colonization of a surface, twitching motility, biofilm formation, aggregation of bacterial cells, which helps in moisture retention and UV tolerance, etc. On the other hand, neither attachment nor pili are indispensable in most cases, but they are apparent epiphytic fitness factors, although very few studies have been performed to confirm this.
Type IV
Pili Type IV pili that also go by several alternative names (type 4 fimbriae, common pili, bundle-forming pili, etc.) are produced by a great number of gram-negative bacteria, including, as it seems, most gram-negative plant pathogens. The genes for biogenesis of type IV pili show homology to those of type II secretion (general secretory pathway), and although many bacteria carry the genes required for pilus biogenesis, pilus production has not always been observed.
The fact that pili have not been observed in the case of a certain strain does not signify a lack of pili, since a clear phenotype is not always associated with the presence of pili. The pili are often expressed in very low numbers and have in some cases been shown to be inducible in certain specific conditions. In the case of Pseudomonas syringae this type of pili was observed at an early stage as the receptors for phage f6, but only later their identity was confirmed as type IV pili. At that point, it was already known that the pili also have a function in promoting attachment of bacteria to plant leaf surfaces, in aggregating bacteria to form pellicles on the surface of stationary cultures and clusters, which influences the UV resistance of the bacteria, and possibly also in the formation of biofilms on plant surfaces.
No plant surface receptor for the type IV pilus has been isolated, and it is possible that the attachment is merely a result of hydrophobic interaction between the pilus and the leaf cuticle. These pili are not required for pathogenesis as such, since nonpiliated mutant strains still cause HR in nonsusceptible plants and disease symptoms in susceptible plants when infiltrated into the plant tissue. The pili are, however, apparently epiphytic fitness factors, the effect of which is seen in a situation of competition. Nonpiliated bacterial mutants initiate colonization less efficiently than the parental wild-type strain, and this difference is maintained in a situation of competition. In the absence of competition, even the nonpiliated strain reaches high population densities but is more sensitive to dislocation by flushing water.
Fimbriae: Type 1, Type 3, etc.
Many bacteria of the Enterobacteriaceae family form type 1 and type 3 fimbriae, which have been shown to mediate the attachment of bacterial cells to target tissues in the case of animal diseases. Also, in some plant-pathogenic and other plant-associated bacteria, these fimbriae have been observed, but a direct correlation to pathogenicity or virulence is in most cases missing. Erwinia rhapotici produces fimbriae with specific adherence to N-acetyllactoseamine. The bacterium infects rhubarb leaves but also infects wheat, causing pink grains to be formed. Receptor analogs, particularly N-acetyllactoseamine, were able to inhibit bacterial attachment and grain coloration. The true pathogenic and ecological role of the Enterobacterial fimbriae/pili is, however, unclear.
Attachment Inside the Leaf Tissue: The Hrp Pilus
The Hrp pathogenicity island (PAI) is essential for the pathogenesis of hypersensitivity reaction in the case of many biotrophic plants and pathogenic bacteria species such as P. syringae. Recently Hrp genes were also shown to play a role in the interaction of the necrotrophic pathogen Erwinia carotovora ssp. carotovora with its host and the Hrp-PAI has been observed in many additional bacterial species. The Hrp pathogenicity island contains a type III secretion system with homology to the gene cluster required for flagellar synthesis.
Type III protein secretion is widespread among gram-negative pathogens of both animals and plants. Apparently, the products of so-called avirulence genes and some pathogenicity determinants are translocated from the bacterial cell, possibly directly inside the plant cell cytoplasm. An idea of how this might take place emerged when the Hrp pilus was discovered. Recently the pilus was shown to grow by the addition of pilin (HrpA) monomers at the pilus tip and to function as a fountain secreting Harpin (HrpZ) at the growing tip.
The pilus grows through the plant cell wall until it reaches and possibly penetrates the plasma membrane, enabling pathogenicity determinants to enter the plant cell. At this point, the bacteria are attached and directly linked to the plant cell, and although the Hrp pilus–mediated attachment is not necessarily physically strong, it is required for the proper functioning of the interaction. In R. solanacearum bacterial cells were observed to attach in a polar manner to plant cells, but this form of attachment was not dependent on Hrp pili. Instead, a different pilus/fimbria might be involved. Whether foliar pathogens depend on a primary or additional adhesin for successful Hrp pilus–mediated interaction is not known.
The results obtained with the Hrp pilus show that proteins can be transported in the lumen of the apparently hollow pilus. It is likely that in order to fit through the tube that the pilus constitutes, the protein has to be unfolded. The pilin itself, HrpA, possibly folds as it emerges at the pilus tip and attaches to the previous HrpA residue. Whether a specific link to a plant receptor is formed when the pilus tip reaches the plant cell membrane is presently not known, but other bacterial proteins, such as the harp, may also play a role here.
Attachment of Agrobacterium
Attachment to plant cells is a necessary and early step in the disease of dicot plants caused by Agrobacterium. The bacterium colonizes the rhizosphere but attaches also to wounded aerial plant tissues. Chemotactic motility towards wounds is followed by attachment, the first step of which apparently is dependent on one or more bacterial cell surface protein(s). Several types of mutants with impaired binding have been observed, but it appears that in some cases the effect is pleiotropic and can be overcome by changing the incubation conditions, such as temperature or osmolarity. After initial binding, cellulose fibrils produced by the bacterium anchor the cells to the wounded plant tissue.
Early binding steps are followed by specific cell-to-cell interactions characteristic for only Agrobacterium. The bacterial cell transfers genetic material directly from the bacterial cell to the cytoplasm of the plant cell through a direct link formed by the bacterium. The molecular and structural details of how the transfer takes place is still partly obscure; whether transfer requires direct contact between participating cells, which are brought together by retraction of the Agrobacterium Tpilus, or whether the translocation of the protein-DNA complex is mediated by the elongated pilus is still a matter of controversy. The Agrobacterium type IV secretion system, showing homology to the F-conjugation system, shares no homology to the Hrp system (type III secretion) of P. syringae and other pathogens, but these two secretion systems do show mechanistic similarities.
Confocal laser scanning micrograph of bacteria inoculated by spraying onto plant leaves followed by live/dead staining of the bacterial cells. The bar denotes 50 mm. Living bacteria appear green against a background of dark red fluorescing leaf tissue. Brighter red spots indicate the presence of chloroplasts. A: Pseudomonas syringae pv. tomato DC3000 scattered over the lower surface of a tomato leaf. The guard cells and opening of a stomate is seen in the middle of the picture. Plant cell outlines cannot be distinguished, but in equivalent scanning electron micrographs (not shown) bacteria can be seen in crevices between neighboring cells and occasionally clustering at and on top of stomata as well as attached to trichomes. B: Erwinia carotovora subsp. carotovora cells aggregating onto the trichome of an Arabidopsis leaf.
CONCLUSION
Bacterial attachment to leaf surfaces is not an absolutely required trait, shown by the fact that plant-associated bacteria can colonize and plant-pathogenic bacteria can cause disease even if genes normally associated with attachment are mutated. The testing scenarios in these cases are, however, never natural. The bacteria are applied alone and usually in large quantities, whereas in natural conditions where any inoculum is likely to be mixed