Plants appear to have two types of active defenses, a broad-spectrum basal system and a system controlled by R-genes providing stronger resistance to some pathogens that break the basal defense. However, it is unknown if the systems are separate entities. Therefore, we analyzed proteins from leaves of the dry bean crop plant Phaseolus vulgaris using a high-throughput liquid chromatography tandem mass spectrometry method. By statistically comparing the amounts of proteins detected in a single plant variety that is susceptible or resistant to infection, depending on the strains of a rust fungus introduced, we defined basal and R-gene-mediated plant defenses at the proteomic level. The data reveal that some basal defense proteins are potential regulators of a strong defense weakened by the fungus and that the R-gene modulates proteins similar to those in the basal system. The results satisfy a new model whereby R-genes are part of the basal system and repair disabled defenses to reinstate strong resistance. Molecular & Cellular Proteomics 8:19 -31, 2009.

BackgroundPhaseolus vulgaris (common bean) is the second most important legume crop in the world after soybean. Consequently, yield losses due to fungal infection, like Uromyces appendiculatus (bean rust), have strong consequences. Several resistant genes were identified that confer resistance to bean rust infection. However, the downstream genes and mechanisms involved in bean resistance to infection are poorly characterized.ResultsA subtractive bean cDNA library composed of 10,581 unisequences was constructed and enriched in sequences regulated by either bean rust race 41, a virulent strain, or race 49, an avirulent strain on cultivar Early Gallatin carrying the resistance gene Ur-4. The construction of this library allowed the identification of 6,202 new bean ESTs, significantly adding to the available sequences for this plant. Regulation of selected bean genes in response to bean rust infection was confirmed by qRT-PCR. Plant gene expression was similar for both race 41 and 49 during the first 48 hours of the infection process but varied significantly at the later time points (72–96 hours after inoculation) mainly due to the presence of the Avr4 gene in the race 49 leading to a hypersensitive response in the bean plants. A biphasic pattern of gene expression was observed for several genes regulated in response to fungal infection.ConclusionThe enrichment of the public database with over 6,000 bean ESTs significantly adds to the genomic resources available for this important crop plant. The analysis of these genes in response to bean rust infection provides a foundation for further studies of the mechanism of fungal disease resistance. The expression pattern of 90 bean genes upon rust infection shares several features with other legumes infected by biotrophic fungi. This finding suggests that the P. vulgaris-U. appendiculatus pathosystem could serve as a model to explore legume-rust interaction.

We are interested in learning more about the proteome of Uromyces appendiculatus, the fungus that causes common bean rust. Knowledge of the proteins that differentiate life-cycle stages and distinguish infectious bodies such as uredospores, germlings, appressoria, and haustoria may be used to define host-pathogen interactions or serve as targets for chemical inhibition of the fungus. We have used 2-D nanoflowLC-MS/MS to identify more than 400 proteins from asexual uredospores. A majority of the proteins appear to have roles in protein folding or protein catabolism. We present a model by which an abundance of heat shock proteins and translation elongation factors may enhance a spore's ability to survive environmental stresses and rapidly initiate protein production upon germination.

Infection with the protozoan parasite Leishmania amazonensis can cause diverse clinical forms of leishmaniasis. Immunization with purified P4 nuclease protein has been shown to elicit a protective response in mice challenged with L. amazonensis and L. pifanoi. To explore the potential of a DNA-based vaccine, we tested the L. amazonensis gene encoding P4 nuclease as well as adjuvant constructs encoding murine interleukin-12 (IL-12) and L. amazonensis HSP70. Susceptible BALB/c mice were immunized with the DNA encoding P4 alone, P4/IL-12, or P4/HSP70 prior to challenge with L. amazonensis promastigotes. Mice given P4/IL-12 exhibited no lesion development and had a 3-to 4-log reduction in tissue parasite burdens compared to controls. This protection corresponded to significant increases in gamma interferon and tumor necrosis factor alpha production and a reduction in parasite-specific immunoglobulin G1, suggesting an enhancement in Th1 responses. Moreover, we immunized mice with the L. amazonensis vaccines to determine if this vaccine regimen could provide cross-protection against a genetically diverse species, L. major. While the P4/HSP70 vaccine led to self-healing lesions, the P4/IL-12 vaccine provided negligible protection against L. major infection. This is the first report of successful use of a DNA vaccine to induce protection against L. amazonensis infection. Additionally, our results indicate that different vaccine combinations, including DNA encoding P4, HSP70, or IL-12, can provide significant protection against both Old World and New World cutaneous leishmaniasis.Leishmaniasis is widespread in over 88 countries. It is estimated that 350 million people live in areas where it is endemic, with 12 million people infected, and that approximately 1.5 million new cases occur each year (65). Current control measures rely on chemotherapy, vector control, and control of reservoir host populations. The chemotherapeutic agents used presently are inadequate, expensive, and often toxic. Due to the existing problems associated with leishmaniasis and the high incidence of infection, the World Health Organization has made it a major goal to develop an effective and affordable vaccine against leishmaniasis.The different Leishmania species cause a broad spectrum of human diseases. L. amazonensis is known to be associated with cutaneous, diffuse cutaneous, and visceral leishmaniasis in South and Central America. The pathological mechanisms responsible for the variable outcomes of infection in humans are not fully understood; however, it is generally agreed that longlasting immunity against reinfection can be developed in cutaneous leishmaniasis patients. Several vaccination trials have demonstrated that killed L. amazonensis can induce protection from natural infection (3,18,42,46,63). However, the efficacy of heat-killed vaccines against Leishmania has been extremely low (36) or highly variable within the same study (47,55). Live parasites have been used as a vaccine strategy, and although they are highly effective in indu...