Indole Alkaloid Production by the Halo Blight Bacterium Treated with the Phytoalexin Genistein.

When Pseudomonas savastanoi pv. phaseolicola, the bacterium that causes halo blight, induces hypersensitive immunity in common bean leaves, salicylic acid and phytoalexins accumulate at the site of infection. Both salicylic acid and the phytoalexin resveratrol exert antibiotic activities and toxicities in vitro, adversely disrupting the P. savastanoi pv. phaseolicola proteome and metabolism and stalling replication and motility. These efficacious properties likely contribute to the cessation of bacterial spread in beans. Genistein is an isoflavonoid phytoalexin that also accumulates during bean immunity, so we tested its antibiotic potential in vitro. Quantitative proteomics revealed that genistein did not induce proteomic changes in P. savastanoi pv. phaseolicola in the same way that salicylic acid or resveratrol did. Rather, a dioxygenase that could function to metabolize genistein was among the most highly induced enzymes. Indeed, high-throughput metabolomics provided direct evidence for genistein catabolism. Metabolomics also revealed that genistein induced the bacterium to produce indole compounds, several of which had structural similarity to auxin. Additional mass spectrometry analyses proved that the bacterium produced an isomer of the auxin indole-3-acetic acid but not indole-3-acetic acid proper. These results reveal that P. savastanoi pv. phaseolicola can tolerate bean genistein and that the bacterium likely responds to bean-produced genistein during infection, using it as a signal to increase pathogenicity, possibly by altering host cell physiology or metabolism through the production of potential auxin mimics.

Salicylic Acid and Phytoalexin Induction by a Bacterium that Causes Halo Blight in Beans.

Pseudomonas savastanoi pv. phaseolicola is a bacterium that causes halo blight in beans. Different varieties of beans have hypersensitive resistance to specific races of P. savastanoi pv. phaseolicola. During hypersensitive resistance, also known as effector-triggered immunity (ETI), beans produce hormones that signal molecular processes to produce phytoalexins that are presumed to be antibiotic to bacteria. To shed light on hormone and phytoalexin production during immunity, we inoculated beans with virulent and avirulent races of P. savastanoi pv. phaseolicola. We then used mass spectrometry to measure the accumulation of salicylic acid (SA), the primary hormone that controls immunity in plants, and other hormones including jasmonate, methyljasmonate, indole-3-acetic acid, abscisic acid, cytokinin, gibberellic acid, and 1-aminocyclopropane-1-carboxylic acid. SA, but no other examined hormone, consistently increased at sites of infection to greater levels in resistant beans compared with susceptible beans at 4 days after inoculation. We then monitored 10 candidate bean phytoalexins. Daidzein, genistein, kievitone, phaseollin, phaseollidin, coumestrol, and resveratrol substantially increased alongside SA in resistant beans but not in susceptible beans. In vitro culture assays revealed that SA, daidzein, genistein, coumestrol, and resveratrol inhibited P. savastanoi pv. phaseolicola race 5 culture growth. These results demonstrate that these phytoalexins may be regulated by SA and work with SA during ETI to restrict bacterial replication. This is the first report of antibiotic activity for daidzein, genistein, and resveratrol to P. savastanoi pv. phaseolicola. These results improve our understanding of the mechanistic output of ETI toward this bacterial pathogen of beans.

Bacterial Immobilization and Toxicity Induced by a Bean Plant Immune System.

Pseudomonas savastanoi pv. phaseolicola causes halo blight disease in the common bean Phaseolus vulgaris. The bacterium invades the leaf apoplast and uses a type III secretion system to inject effector proteins into a bean cell to interfere with the bean immune system. Beans counter with resistance proteins that can detect effectors and coordinate effector-triggered immunity responses transduced by salicylic acid, the primary defense hormone. Effector-triggered immunity halts bacterial spread, but its direct effect on the bacterium is not known. In this study, mass spectrometry of bacterial infections from immune and susceptible beans revealed that immune beans inhibited the accumulation of bacterial proteins required for virulence, secretion, motility, chemotaxis, quorum sensing, and alginate production. Sets of genes encoding these proteins appeared to function in operons, which implies that immunity altered the coregulated genes in the bacterium. Immunity also reduced amounts of bacterial methylglyoxal detoxification enzymes and their transcripts. Treatment of bacteria with salicylic acid, the plant hormone produced during immunity, reduced bacterial growth, decreased gene expression for methylglyoxal detoxification enzymes, and increased bacterial methylglyoxal concentrations in vitro. Increased methylglyoxal concentrations reduced bacterial reproduction. These findings support the hypothesis that plant immunity involves the chemical induction of adverse changes to the bacterial proteome to reduce pathogenicity and to cause bacterial self-toxicity.

The Proteomics of Resistance to Halo Blight in Common Bean.

Halo blight disease of beans is caused by a gram-negative bacterium, Pseudomonas syringae pv. phaseolicola. The disease is prevalent in South America and Africa and causes crop loss for indigent people who rely on beans as a primary source of daily nutrition. In susceptible beans, P. syringae pv. phaseolicola causes water-soaking at the site of infection and produces phaseolotoxin, an inhibitor of bean arginine biosynthesis. In resistant beans, P. syringae pv. phaseolicola triggers a hypersensitive response that limits the spread of infection. Here, we used high-throughput mass spectrometry to interrogate the responses to two different P. syringae pv. phaseolicola isolates on a single line of common bean, Phaseolus vulgaris PI G19833, with a reference genome sequence. We obtained quantitative information for 4,135 bean proteins. A subset of 160 proteins with similar accumulation changes during both susceptible and resistant reactions included salicylic acid responders EDS1 and NDR1, ethylene and jasmonic acid biosynthesis enzymes, and proteins enabling vesicle secretion. These proteins revealed the activation of a basal defense involving hormonal responses and the mobilization of extracellular proteins. A subset of 29 proteins specific to hypersensitive immunity included SOBIR1, a G-type lectin receptor-like kinase, and enzymes needed for glucoside and phytoalexin production. Virus-induced gene silencing revealed that the G-type lectin receptor-like kinase suppresses bacterial infection. Together, the results define the proteomics of disease resistance to P. syringae pv. phaseolicola in beans and support a model whereby the induction of hypersensitive immunity reinstates defenses targeted by P. syringae pv. phaseolicola.

Benzothiadiazole Conditions the Bean Proteome for Immunity to Bean Rust.

The common bean rust fungus reduces harvests of the dry, edible common bean. Natural resistance genes in the plant can provide protection until a fungal strain that breaks resistance emerges. In this study, we demonstrate that benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) sprayed on susceptible beans induces resistance to common bean rust. Protection occurred as soon as 72 h after treatment and resulted in no signs of disease 10 days after inoculation with rust spores. By contrast, the susceptible control plants sustained heavy infections and died. To understand the effect BTH has on the bean proteome, we measured the changes of accumulation for 3,973 proteins using mass spectrometry. The set of 409 proteins with significantly increased accumulation in BTH-treated leaves included receptor-like kinases SOBIR1, CERK1, and LYK5, which perceive pathogens, and EDS1, a regulator of the salicylic acid defense pathway. Other proteins that likely contributed to resistance included pathogenesis-related proteins, a full complement of enzymes that catalyze phenylpropanoid biosynthesis, and protein receptors, transporters, and enzymes that modulate other defense responses controlled by jasmonic acid, ethylene, brassinosteroid, abscisic acid, and auxin. Increases in the accumulation of proteins required for vesicle-mediated protein secretion and RNA splicing occurred as well. By contrast, more than half of the 168 decreases belonged to chloroplast proteins and proteins involved in cell expansion. These results reveal a set of proteins needed for rust resistance and reaffirm the utility of BTH to control disease by amplifying the natural immune system of the bean plant.

A Proteomic Network for Symbiotic Nitrogen Fixation Efficiency in Bradyrhizobium elkanii.

Rhizobia colonize legumes and reduce N2 to NH3 in root nodules. The current model is that symbiotic rhizobia bacteroids avoid assimilating this NH3. Instead, host legume cells form glutamine from NH3, and the nitrogen is returned to the bacteroid as dicarboxylates, peptides, and amino acids. In soybean cells surrounding bacteroids, glutamine also is converted to ureides. One problem for soybean cultivation is inefficiency in symbiotic N2 fixation, the biochemical basis of which is unknown. Here, the proteomes of bacteroids of Bradyrhizobium elkanii USDA76 isolated from N2 fixation-efficient Peking and -inefficient Williams 82 soybean nodules were analyzed by mass spectrometry. Nearly half of the encoded bacterial proteins were quantified. Efficient bacteroids produced greater amounts of enzymes to form Nod factors and had increased amounts of signaling proteins, transporters, and enzymes needed to generate ATP to power nitrogenase and to acquire resources. Parallel investigation of nodule proteins revealed that Peking had no significantly greater accumulation of enzymes needed to assimilate NH3 than Williams 82. Instead, efficient bacteroids had increased amounts of enzymes to produce amino acids, including glutamine, and to form ureide precursors. These results support a model for efficient symbiotic N2 fixation in soybean where the bacteroid assimilates NH3 for itself.

Protection Against Common Bean Rust Conferred by a Gene-Silencing Method.

Rust disease of the dry bean plant, Phaseolus vulgaris, is caused by the fungus Uromyces appendiculatus. The fungus acquires its nutrients and energy from bean leaves using a specialized cell structure, the haustorium, through which it secretes effector proteins that contribute to pathogenicity by defeating the plant immune system. Candidate effectors have been identified by DNA sequencing and motif analysis, and some candidates have been observed in infected leaves by mass spectrometry. To assess their roles in pathogenicity, we have inserted small fragments of genes for five candidates into Bean pod mottle virus. Plants were infected with recombinant virus and then challenged with U. appendiculatus. Virus-infected plants expressing gene fragments for four of five candidate effectors accumulated lower amounts of rust and had dramatically less rust disease. By contrast, controls that included a fungal gene fragment for a septin protein not expressed in the haustorium died from a synergistic reaction between the virus and the fungus. The results imply that RNA generated in the plant moved across the fungal haustorium to silence effector genes important to fungal pathogenicity. This study shows that four bean rust fungal genes encode pathogenicity determinants and that the expression of fungal RNA in the plant can be an effective method for protecting bean plants from rust.

Putative Rust Fungal Effector Proteins in Infected Bean and Soybean Leaves.

The plant-pathogenic fungi Uromyces appendiculatus and Phakopsora pachyrhizi cause debilitating rust diseases on common bean and soybean. These rust fungi secrete effector proteins that allow them to infect plants, but their effector repertoires are not understood. The discovery of rust fungus effectors may eventually help guide decisions and actions that mitigate crop production loss. Therefore, we used mass spectrometry to identify thousands of proteins in infected beans and soybeans and in germinated fungal spores. The comparative analysis between the two helped differentiate a set of 24 U. appendiculatus proteins targeted for secretion that were specifically found in infected beans and a set of 34 U. appendiculatus proteins targeted for secretion that were found in germinated spores and infected beans. The proteins specific to infected beans included family 26 and family 76 glycoside hydrolases that may contribute to degrading plant cell walls. There were also several types of proteins with structural motifs that may aid in stabilizing the specialized fungal haustorium cell that interfaces the plant cell membrane during infection. There were 16 P. pachyrhizi proteins targeted for secretion that were found in infected soybeans, and many of these proteins resembled the U. appendiculatus proteins found in infected beans, which implies that these proteins are important to rust fungal pathology in general. This data set provides insight to the biochemical mechanisms that rust fungi use to overcome plant immune systems and to parasitize cells.

Expression of a synthetic rust fungal virus cDNA in yeast.

Mycoviruses are viruses that infect fungi. Recently, mycovirus-like RNAs were sequenced from the fungus Phakopsora pachyrhizi, the causal agent of soybean rust. One of the RNAs appeared to represent a novel mycovirus and was designated Phakopsora pachyrhizi virus 2383 (PpV2383). The genome of PpV2383 resembles Saccharomyces cerevisiae virus L-A, a double-stranded (ds) RNA mycovirus of yeast. PpV2383 encodes two major, overlapping open reading frames with similarity to gag (capsid protein) and pol (RNA-dependent RNA polymerase), and a -1 ribosomal frameshift is necessary for the translation of a gag-pol fusion protein. Phylogenetic analysis of pol relates PpV2383 to members of the family Totiviridae, including L-A. Because the obligate biotrophic nature of P. pachyrhizi makes it genetically intractable for in vivo analysis and because PpV2383 is similar to L-A, we synthesized a DNA clone of PpV2383 and tested its infectivity in yeast cells. PpV2383 RNA was successfully expressed in yeast, and mass spectrometry confirmed the translation of gag and gag-pol fusion proteins. There was, however, no production of PpV2383 dsRNA, the evidence of viral replication. Neither the presence of endogenous L-A nor the substitution of the 5' and 3' untranslated regions with those from L-A was sufficient to rescue replication of PpV2383. Nevertheless, the proof of transcription and translation from the clone in vivo are steps toward confirming that PpV2383 is a mycovirus. Further development of a surrogate biological system for the study of rust mycoviruses is necessary, and such research may facilitate biological control of rust diseases.

Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing.

Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.

Nuclear proteomic changes linked to soybean rust resistance.

Soybean rust, caused by the fungus Phakopsora pachyrhizi, is an emerging threat to the US soybean crop. In an effort to identify proteins that contribute to disease resistance in soybean we compared a susceptible Williams 82 cultivar to a resistant Williams 82 inbred isoline harboring the Rpp1 resistance gene (R-gene). Approximately 4975 proteins from nuclear preparations of leaves were detected using a high-throughput liquid chromatography-mass spectrometry method. Many of these proteins have predicted nuclear localization signals, have homology to transcription factors and other nuclear regulatory proteins, and are phosphorylated. Statistics of summed spectral counts revealed sets of proteins with differential accumulation changes between susceptible and resistant plants. These protein accumulation changes were compared to previously reported gene expression changes and very little overlap was found. Thus, it appears that numerous proteins are post-translationally affected in the nucleus after infection. To our knowledge, this is the first indication of large-scale proteomic change in a plant nucleus after infection. Furthermore, the data reveal distinct proteins under control of Rpp1 and show that this disease resistance gene regulates nuclear protein accumulation. These regulated proteins likely influence broader defense responses, and these data may facilitate the development of plants with improved resistance.

Generation of Phaseolus vulgaris ESTs and investigation of their regulation upon Uromyces appendiculatus infection.

BACKGROUND: Phaseolus 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. RESULTS: A 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. CONCLUSION: The 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.

Quantitative proteomic analysis of bean plants infected by a virulent and avirulent obligate rust fungus.

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.

Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences.

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.

Protein accumulation in the germinating Uromyces appendiculatus uredospore.

Uromyces appendiculatus is a rust fungus that causes disease on beans. To understand more about the biology of U. appendiculatus, we have used multidimensional protein identification technology to survey proteins in germinating asexual uredospores and have compared this data with proteins discovered in an inactive spore. The relative concentrations of proteins were estimated by counting the numbers of tandem mass spectra assigned to peptides for each detected protein. After germination, there were few changes in amounts of accumulated proteins involved in glycolysis, acetyl Co-A metabolism, citric acid cycle, ATP-coupled proton transport, or gluconeogenesis. Moreover, the total amount of translation elongation factors remained high, supporting a prior model that suggests that germlings acquire protein translation machinery from uredospores. However, germlings contained a higher amount of proteins involved in mitochondrial ADP:ATP translocation, which is indicative of increased energy production. Also, there were more accumulating histone proteins, pointing to the reorganization of the nuclei that occurs after germination prior to appressorium formation. Generally, these changes are indicative of metabolic transition from dormancy to germination and are supported by cytological and developmental models of germling growth.

Shotgun identification of proteins from uredospores of the bean rust Uromyces appendiculatus.

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.