Bacterial Speck Disease of Tomato: An Insight into Host-Bacteria Interaction
Department of Plant Pathology
University of Minnesota
St. Paul, MN 55108, USA
Pseudomonas syringae pv. tomato (Pst) is a common pathogen of tomato which causes bacterial speck disease. This disease serves as a useful model for studying the interactions of microbial pathogens and plants. Most gram-negative bacteria, including Pst, have type III secretion system (TTSS). Encoded by hrp gene clusters, the TTSS is used to deliver effector proteins into the host cytosol. The hrp genes also control the expression of the avirulence genes (avr). One Avr protein, AvrPto, functions as ligand to elicit a hypersensitive response (HR) in the tomato plant after recognition by the protein encoded by the host resistance gene, Pto. The AvrPto- Pto interaction is the most widely studied systems. It has been discovered that Pto is linked with Fen, the gene responsible for susceptibility to an organophosphate insecticide, fenthion. Functioning of Pto requires another gene called Prf, which lies embedded in Pto. Though the system is well characterized, several aspects are still not understood. With the availability of completed genome sequence of Pst and the full sequence of tomato expected in the future, we may anticipate that our understanding of the mechanisms of this host-pathogen interaction to be improved.
Biological aspect of interaction
P seudomonas syringae pv. tomato (Pst) causes bacterial speck disease of tomato wherever tomatoes are grown (8). Primary sites of infection are stomata, the bases of leaf trichomes and wounds (2). Following infection, bacteria multiply in the leaf interior by forming microcolonies in close physical association with the cell wall of host mesophyll cells (15). Once the host intercellular spaces are filled with Pst, host cells are polymerized and degenerate (37). Disease symptoms may be evident on all aboveground plant parts, though immature tissues are the most susceptible. Symptoms on leaves are often indistinct. Leaf spots are dark and round, and often have a discrete halo. As the disease progresses, lesions may extend into the petiole and stems. On fruit, the disease initially appears as small black spots of 1/8 - 1/4 inch diameter with distinct margins. These small spots are superficial, do not rupture the epidermis and will not develop into soft rot. Lesions on fruit are sometimes surrounded by an area that is slow to ripen. When fruits are infected early, the spots may cause pit-like distortions because the host tissues within lesions grow slower than unaffected tissue. Mature fruits are resistant to Pst infection as a result of their high acidity (8,38,42). Formation of halo in speck symptoms is due to the toxin, coronatine (COR), produced by Pst (3). Serious disease outbreaks, though rare, are favored by high leaf wetness, cool temperature and cultural practices that allow bacteria to be disseminated between hosts (26). Though the economic impact of disease is minimal, it is important from the point of view of scientific study of host- pathogen interactions. When Pst infects susceptible tomato plants it causes typical disease symptoms. In contrast, infection of a resistant plant is restricted by the localized death of the cells at the site of infection known as the hypersensitive response (HR). The HR is generally microscopic but when it occurs over larger areas, becomes macroscopic. In addition, it has been reported that Pst can cause HR in non-host plants, which has been described as type II non-host resistance and shares some mechanistic similarities to the HR defense mediated by resistance genes (13,17).
Evidence for Communication between Microbe and Plant or vise versa-
The first action required for successful infection by pathogen is entrance into the host plant. The stomatal entry is important for Pst and is aided by the toxin, COR (36). Under normal conditions plant stomata close, in response to signals perceived by guard cells in the presence of live bacteria, to limit bacterial entry. Stomata close upon detection of pathogen associated molecular patterns (PAMPs). PAMPs are mostly lipopolysaccharides (LPS) and flagellin (flg22) which are identified by an unknown immune receptor and flagellin receptor, FLS2, respectively. The pathway for stomatal closure involves triggering of the salicylic acid (SA) and abscisic acid (ABA) signaling pathways (15). However COR produced by Pst in the apoplast or on the leaf surfaces re-opens closed stomata thereby increasing the liklihood of Pst invasion. Malic acid and citric acid, with minor contributions from shikimic acid and quinic acid, released from the plant tissues induce COR production (16). The COR promotes stomatal reopening through the E3 ligase subunit COI1, a key component of jasmonic acid (JA) signaling pathway. How Pst recognizes open and closed stomata is not clear. It is speculated that Pst movement and detection of stomatal status is chemotaxic due to nutrients released from interior of leaves through open stomata. Thus COR is an important virulence factor, which promotes colonization of host tissue by suppression of PAMPs induced early defense response in tomato (15,20,21).
Pst and other plant pathogenic bacteria have evolved a variety of virulence factors to avoid or supress host defenses and colonize plants successfully. One such virulence factor is the hypersensitive response and pathogenicity (hrp)-gene-encoded type III secretion system, (TTSS; see detail in molecular biology and genetics section). The TTSS is used by bacteria to inject a large number of virulence effector proteins into the host cell. Once Pst is inside the plant’s apoplast, it needs signals for the induction of the hrp gene cluster. Mostly hrp gene expression is under the control of signals from the plant, and physiological and environmental conditions. In a study, when different hrp genes were expressed under varying conditions in vitro, optimum expression of all hrp genes were under conditions that simulate the apoplastic condition (low pH and limited nutrient condition: ie hypo-osmotic environment). Similarly, hrpL and hrpRS, members of hrp gene family, were actively expressed only in host plant leaves (27). Thus, once Pst are in the apoplast, the osmotic pressure, nutrient and pH condition of apoplast and some other signals from the plant induce the expression of hrp genes.
Physiological Alterations in the Participants
Several genes are induced both in Pst and tomato plants after infection. Especially, after the production of different effector proteins through the TTSS, there is up- and down-regulation of several genes that alter the physiology of host cells and induce the defense mechanisms.
These changes include, cell wall callus deposition, lignification of cell wall, oxidative burst, increase in the cell protectants, up-regulation of anthocyanin biosynthesis, JA and alkaloid biosynthesis, senescence, upregulation of defense related genes and ultimately the HR. Similarly, photosynthesis related activity, cell osmotic pressure related genes, and some protein kinases are down-regulated (Fig.1) (25).
Infection of tomato plants by Pst also alters the nitrogen metabolism of the host plant. Similar to abiotic stresses, Pst infection leads to reduced nitrogen assimilation in the infected cells. This has been shown by reduced expression of the primary nitrogen assimilation genes, GS2 and Nia (24). Similarly, nitric oxide (NO), an important component for the induction of defense response, has been shown to be produced in a hrp dependent manner in host cells after Pst infection. NO production was preceded by the generation of hydrogen peroxide (H2O2) (22). However, NO production leading to basal defense has also been shown to occur in a hrp independent manner (36). Accumulation of a cytosolic glutamine synthetase (GS1) isoform, very similar to the GS1 isoform induced in tobacco leaves during senescence, was also observed (24). This indicates that the physiological processes induced during Pst infection are similar to those involved in natural senescence. It is interesting fact that several pathogen-related (PR) defensive proteins are expressed also in natural senescence. Wright and Beattie (40) observed that there is change in osmotic pressure of host cells after Pst infection. Pst encountered lower potential in incompatible reaction than in compatible reaction in tobacco plants. Thus, a reduction in the water potential of cells may restrict the endophytic growth of Pst. All these changes, decrease in water potential, reduction in nitrogen assimilation, and physiology similar to natural senescence are possibly linked to the HR. There is a possibility that Pst changes the host cell physiology through production of the phytohormone auxin. It has been indicated by induction of a gene, iaaL, by hrpL which encodes indoleacetate-lysine ligase capable of producing an inactive form of auxin and indoleacitic acid (IAA) in Pst DC3000 (35).
Host cell physiology can change even before the translocation of effectors proteins into the host cytosol. As mentioned earlier, the TTSS serves as a virulence factor for Pst. However, TTSS alone does not appear to be sufficient for bacteria to cause disease. Thus, a variety of virulence factors, such as COR, are necessary for full virulence. These virulence factors affect host cell by altering physiology. For example due to COR, Pst is able to reopen the stomata that are closed in response to the PAMPs through JA signaling pathway.
When a gene resistance to P . syringae pv. to mato (Pto) was transferred to susceptible tomato plants through breeding, there was an unintended consequence. Resistant tomato plants were found to be susceptible to organophosphorus insecticide fenthion (29). Susceptibility to fenthion resulted into HR similar to the HR produced Pto mediated resistace in tomato plants (28). With genetic study it was discovered that Pto resistance and fenthion sensitivity are
encoded by two different genes, Pto and Fen respectively. The Fen gene is a Pto homolog but not involved in AvrPto recognition (28,29). The fenthion susceptibility trait is widely used for breeding resistance to bacterial speck of tomato (25).
* Author current address: Small Grains Pathology Lab, Department of Plant Science, South Dakota State University Brookings, SD 57007, USA