The influence of genetics,defensive chemistry and the fungal microbiome on disease outcome in whitebark pine trees

The invasive fungal pathogen Cronartium ribicola infects and kills whitebark pine (Pinus albicaulis) throughout western North America. Whitebark pine has been proposed for listing under the Endangered Species Act in the USA, and the loss of this species is predicted to have severe impacts on ecosystem composition and function in high‐elevation forests. Numerous fungal endophytes live inside whitebark pine tissues and may influence the severity of C. ribicola infection, either directly by inhibition of pathogen growth or indirectly by the induction of chemical defensive pathways in the tree. Terpenes, a form of chemical defence in pine trees, can also influence disease. In this study, we characterized fungal endophyte communities in whitebark pine seedlings before and after experimental inoculation with C. ribicola, monitored disease progression and compared fungal community composition in susceptible vs. resistant seedlings in a common garden. We analysed the terpene composition of these same seedlings. Seed family identity or maternal genetics influenced both terpenes and endophyte communities. Terpene and endophyte composition correlated with disease severity, and terpene concentrations differed in resistant vs. susceptible seedlings. These results suggest that the resistance to C. ribicola observed in natural whitebark pine populations is caused by the combined effects of genetics, endophytes and terpenes within needle tissue, in which initial interactions between microbes and hosts take place. Tree genotype, terpene and microbiome combinations associated with healthy trees could help to predict or reduce disease severity and improve outcomes of future tree breeding programmes.     

Proteome Analysis of Pathogen-Responsive Proteins from Apple Leaves Induced by the Alternaria Blotch Alternaria alternata

Understanding the defence mechanisms used by apple leaves against Alternaria alternate pathogen infection is important for breeding purposes. To investigate the ultrastructural differences between leaf tissues of susceptible and resistant seedlings, in vitro inoculation assays and transmission electron microscopy (TEM) analysis were conducted with two different inoculation assays. The results indicated that the resistant leaves may have certain antifungal activity against A. alternate that is lacking in susceptible leaves. To elucidate the two different host responses to A. alternate infection in apples, the proteomes of susceptible and resistant apple leaves that had or had not been infected with pathogen were characterised using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS). MS identified 43 differentially expressed proteins in two different inoculation assays. The known proteins were categorised into 5 classes, among these proteins, some pathogenesis-related (PR) proteins, such as beta-1,3-glucanase, ascorbate peroxidase (APX), glutathione peroxidase (GPX) and mal d1, were identified in susceptible and resistant hosts and were associated with disease resistance of the apple host. In addition, the different levels of mal d1 in susceptible and resistant hosts may contribute to the outstanding anti-disease properties of resistant leaves against A. alternate. Taken together, the resistance mechanisms of the apple host against A. alternate may be a result of the PR proteins and other defence-related proteins. Given the complexity of the biology involved in the interaction between apple leaves and the A. alternate pathogen, further investigation will yield more valuable insights into the molecular mechanisms of suppression of the A. alternate pathogen. Overall, we outline several novel insights into the response of apple leaves to pathogen attacks. These findings increase our knowledge of pathogen resistance mechanisms, and the data will also promote further investigation into the regulation of the expression of these target proteins.     

Proteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer)

Herbivorous insects can cause severe cellular changes to plant foliage following infestations, depending on feeding behaviour. Here, a proteomic study was conducted to investigate the influence of green peach aphid (Myzus persicae Sulzer) as a polyphagous pest on the defence response of Arabidopsis thaliana (L.) Heynh after aphid colony establishment on the host plant (3 days). Analysis of about 574 protein spots on 2‐DE gels revealed 31 differentially expressed protein spots. Twenty out of these 31 differential proteins were selected for analysis by mass spectrometry. In 12 of the 20 analysed spots, we identified seven and nine proteins using MALDI‐TOF‐MS and LC‐ESI‐MS/MS, respectively. Of the analysed spots, 25% contain two proteins. Different metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defence response and translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis by M. persicae aphids. It provides valuable insights into the complex responses of plants to biological stress, particularly for herbivorous insects with sucking feeding behaviour.     

Identification of Candida albicans exposed surface proteins in vivo by a rapid proteomic approach

We have set up a fast and easy methodology to identify cell-surface proteins in live yeasts. A non-gel proteomic approach was based on a short period of trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS. Candida albicans was used as a model organism and proteins involved in cell wall organization, cell rescue, defense, virulence, transport, protein fate and metabolism were identified. This strategy is a powerful tool to study host–pathogen interactions and to look for potential vaccine candidates and drug targets.     

Identification by 2-D DIGE of apoplastic proteins regulated by oligogalacturonides in Arabidopsis thaliana

Oligogalacturonides (OGs) are elicitors of plant defence responses released from the homogalacturonan of the plant cell wall during the attack by pathogenic micro-organisms. The signalling pathway mediated by OGs remains poorly understood, and no proteins involved in their signal perception and transduction have yet been identified. In order to shed light into the molecular pathways regulated by OGs, a differential proteomic analysis has been carried out in Arabidopsis. Proteins from the apoplastic compartment were isolated and their expression compared between control and OG-treated seedlings. 2-D gels and difference in gel electrophoresis (DIGE) techniques were used to compare control and treated proteomes in the same gel. The analysis of subcellular proteomes from seedlings allowed the identification of novel and low abundance proteins that otherwise remain masked when total cellular extracts are investigated. The DIGE technique showed to be a powerful tool to overcome the high interexperiment variation of 2-D gels. Differentially expressed apoplastic proteins were identified by MS and included proteins putatively involved in recognition as well as proteins whose PTMs are regulated by OGs. Our findings underscore the importance of cell wall as a source of molecules playing a role in the perception of pathogens and provide candidate proteins involved in the response to OGs.     

Differentially Expressed Proteins and Associated Histological and Disease Progression Changes in Cotyledon Tissue of a Resistant and Susceptible Genotype of Brassica napus Infected with Sclerotinia sclerotiorum

Sclerotinia rot caused by Sclerotinia sclerotiorum is one of the most serious diseases of oilseed rape. To understand the resistance mechanisms in the Brassica napus to S. sclerotiorum, comparative disease progression, histological and proteomic studies were conducted of two B. napus genotypes (resistant cv. Charlton, susceptible cv. RQ001-02M2). At 72 and 96 h post inoculation (hpi), lesion size on cotyledons was significantly (P≤0.001) smaller in the resistant Charlton. Anatomical investigations revealed impeded fungal growth (at 24 hpi and onwards) and hyphal disintegration only on resistant Charlton. Temporal changes (12, 24, 48 and 72 hpi) in protein profile showed certain enzymes up-regulated only in resistant Charlton, such as those related to primary metabolic pathways, antioxidant defence, ethylene biosynthesis, pathogenesis related proteins, protein synthesis and protein folding, play a role in mediating defence responses against S. sclerotiorum. Similarly a eukaryotic translation initiation factor 5A enzyme with increased abundance in susceptible RQ001-02M2 and decreased levels in resistant Charlton has a role in increased susceptibility to this pathogen. This is the first time that the expression of these enzymes has been shown to be associated with mediating the defence response against S. sclerotinia in cotyledon tissue of a resistant cultivar of B. napus at a proteomics level. This study not only provides important new insights into the resistance mechanisms within B. napus against S. sclerotiorum, but opens the way for novel engineering of new B. napus varieties that over-express these key enzymes as a strategy to enhance resistance and better manage this devastating pathogen.     

Host plant adaptability and proteomic differences of diverse Rhopalosiphum maidis (Fitch) lineages

Corn leaf aphid Rhopalosiphum maidis (Fitch) can feed on various cereal crops and transmit viruses that may cause serious economic losses. To test the impact of both host plant species and age on R. maidis, as well as the proteomic difference of diverse populations, we first investigated the survival and reproduction of six R. maidis populations (i.e., LF, HF, GZ, DY, BJ, and MS) via a direct observation method in the laboratory on 10 and 50 cm high maize seedlings, and 10 cm high barley seedlings. Then a proteomic approach was implemented to identify the differentially expressed proteins from both aphids and endosymbionts of BJ and MS populations. Results indicated that the BJ population performed significantly better than the others on both barley and 50 cm high maize seedlings, while no population could survive on 10 cm high maize seedlings. The proteomic results demonstrated that the expression levels of myosin heavy chain (muscle isoform X12) (spot 781) and peroxidase (spot 1383) were upregulated, while ATP-dependent protease Hsp 100 (spot 2137) from Hamiltonella defensa and protein SYMBAF (spot 2703) from Serratia symbiotica were downregulated in the BJ population when compared to expression levels of the MS population. We hypothesize that the fatalness observed on 10 cm high maize seedlings may be caused by secondary metabolites that are synthesized by the seedlings and the MS population of R. maidis should be more stress-resistant than the BJ population. Our results also provide insights for understanding the interaction between host plants and aphids.     

Role of gibberellic acid in tomato defence against potato purple top phytoplasma infection

Infection of tomato by potato purple top (PPT) phytoplasma causes disruption of gibberellin (GA) homeostasis in the plant host. Such pathologically‐induced GA deficiency can be partially reversed by exogenous application of GA. This study was designed to explore the role of GA in tomato defence response against phytoplasmal disease, and to determine whether pretreatment with GA would protect healthy tomato seedlings from subsequent phytoplasmal infection and disease development. Our results revealed that, following exogenous GA application and subsequent PPT phytoplasma graft inoculation, there was an apparently coordinated down‐regulation of the gene encoding a key GA signalling component and growth repressor known as DELLA protein (GAI) and up‐regulation of genes involved in salicylic acid (SA) synthesis (ICS1), signalling (NIM1) and downstream defence responses (PRP‐1). Our results also indicated that differential regulation of the above genes was correlated with an increase in activities of defence‐related enzymes β‐1,3‐glucanase and chitinase. The data presented in this communication provide evidence to suggest that GA may act via DELLA and SA signalling pathways to modulate host defence in response to PPT phytoplasma infection. Although the GA pretreatment‐induced defence was not sufficient to prevent a systemic infection, it reduced phytoplasma titre and significantly attenuated disease symptoms. While the actual molecular mechanism underlying the GA‐induced plant defence remains elusive, findings from the current study open new opportunities for in‐depth studies of the functional role of the GA signalling network during defence response against phytoplasmal infection.     

Proteomic profiles of soluble proteins from the esophageal gland in female Meloidogyne incognita

Meloidogyne incognita can infect multiple plant species. Proteins synthesized in the esophageal glands and secreted through the stylet of plant parasitic nematodes play critical roles in the plant-nematode interactions. Female M. incognita live for approximately 15 days, embedded in a host plant, but their esophageal gland proteins have not yet been comprehensively analyzed. In this study, a new bacterium-contamination-resistant method for collecting soluble proteins from esophageal gland cells (SPEGC) of female M. incognita was established. Approximately 5 μg of freeze-dried proteins could be extracted from 150 female M. incognita. Bands of a one-dimensional SDS–polyacrylamide gel were excised after electrophoresis of 20 μg of protein and were analyzed. Two hundred and forty-six proteins from SPEGC of female M. incognita were identified by LC–MS/MS. Gene Ontology analysis suggests that many of the secreted proteins are involved in protein or carbohydrate metabolism and proteolysis. Some of the SPEGC (46.3%) were predicted to be secreted through classical or non-classical secretory pathways. The described method presents a new approach for the identification of proteins stored in SPEGC of an important plant parasitic nematode. This global proteomic profile of SPEGC provides a basis for future studies to elucidate the functions of proteins secreted from female M. incognita on plant responses.     

Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development.     

Echinococcus multilocularis: Proteomic analysis of the protoscoleces by two-dimensional electrophoresis and mass spectrometry

Echinococcus multilocularis is an important parasite that causes human alveolar echinococcosis. Identification and characterization of the proteins encoded by E. multilocularis metacestode might help to understand the complexity of the parasites and their interactions with the host, and to identify new candidates for immunodiagnosis and vaccine development. Here we present a proteomic analysis of E. multilocularis protoscolex (PSC) proteins. The proteins were resolved by 2-DE (pH range 3.5-10), followed by MALDI-TOF MS analysis. Fourteen known Echinococcus proteins were identified, including cytoskeletal proteins, heat shock proteins, metabolic enzymes, 14-3-3 protein, antigen P-29 and calreticulin. To construct a systematic reference map of the immunogenic proteins from E. multilocularis PSC, immunoblot analysis of PSC 2-DE maps was performed. Over 50 proteins spots were detected on immunoblots as antigens and 15 of them were defined. The results showed that cytoskeletal proteins and heat shock proteins were immunodominant antigens in alveolar echinococcosis.     

Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.