Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Zhang CX

PROVIDER: S-EPMC4472855 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Zhang Cai-xia CX Tian Yi Y Cong Pei-hua PH

PloS one 20150618 6

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 la ...[more]

PMID: 26086845

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Project description:Alternaria blotch disease of apple (Malus × domestica Borkh.), caused by the apple pathotype of Alternaria alternata, is one of the most serious fungal diseases to affect apples. To develop an understanding of how apples respond to A. alternata apple pathotype (AAAP) infection, we examined the host transcript accumulation over the period between 0 and 72 h post AAAP inoculation. Large-scale gene expression analysis was conducted of the compatible interaction between "Starking Delicious" apple cultivar and AAAP using RNA-Seq and digital gene expression (DGE) profiling methods. Our results show that a total of 9080 differentially expressed genes (DEGs) were detected (>two-fold and FDR < 0.001) by RNA-Seq. During the early phase of infection, 12 h post inoculation (HPI), AAAP exhibited limited fungal development and little change in the transcript accumulation status (950 DEGs). During the intermediate phase of infection, the period between 18 and 36 HPI, increased fungal development, active infection, and increased transcript accumulation were detected (4111 and 3838 DEGs detected at each time point, respectively). The majority of DEGs were detected by 72 HPI, suggesting that this is an important time point in the response of apples' AAAP infection. Subsequent gene ontology (GO) and pathway enrichment analyses showed that DEGs are predominately involved in biological processes and metabolic pathways; results showed that almost gene associated with photosynthesis, oxidation-reduction were down-regulated, while transcription factors (i.e., WRKY, MYB, NAC, and Hsf) and DEGs involved in cell wall modification, defense signaling, the synthesis of defense-related metabolites, including pathogenesis-related (PRs) genes and phenylpropanoid/cyanoamino acid /flavonoid biosynthesis, were activated during this process. Our study also suggested that the cell wall defensive vulnerability and the down-regulation of most PRs and HSP70s in "Starking Delicious" following AAAP infection might interpret its susceptible to AAAP.

Project description:Apple Alternaria blotch disease, caused by Alternaria alternata (Fr.) Keissl, is one of the most famous leaf diseases. When the disease is prevalent, it causes leaf abscission and influences the formation of flower buds and photosynthesis. Therefore, a simple, rapid, high-specificity and sensitivity method for monitoring infected leaves at early developmental stages is urgently needed, so that the occurrence and expansion of A. alternata can be controlled in time. In our research, a rapid, specific and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect A. alternata within 60 min. Six primers of LAMP detection can only specifically amplify the aapg-1 gene in A. alternata but not in four other important fungi in apples. The aapg-1 gene encodes endopolygalacturonase in A. alternata, and there are significant differences among different species. Thus, it was applied as the target for LAMP primers. Compared to conventional PCR detection, our LAMP method had the same sensitivity as that of detecting as little as 1 fg of pure genomic DNA of A. alternata. When leaves were inoculated with A. alternata conidia, LAMP detected 1 × 102 conidia/mL as the minimum concentration. However, the traditional tissue isolation and identification method only isolated A. alternata from leaves inoculated with 1 × 105 and 1 × 106 conidia/mL, indicating that the LAMP method was more sensitive than the traditional tissue isolation and identification method for A. alternata before symptoms. Further tests also indicated that LAMP detection was more accurate and sensitive than the traditional tissue isolation and identification method for A. alternata in leaves with the Alternaria blotch symptom collected from the field. Our results showed that the LAMP-targeting the aapg-1 gene has the advantages of high sensitivity, specificity and simplicity and can be used for rapid detection and early monitoring of A. alternata in the field. LAMP is instructive for us to effectively prevent and control apple Alternaria blotch disease.

Project description:The fungal genus Alternaria is a pan-global pathogen of > 100 crops, and is associated with the globally expanding Alternaria leaf blotch in apple (Malus x domestica Borkh.) which leads to severe leaf necrosis, premature defoliation, and large economic losses. Up to date, the epidemiology of many Alternaria species is still not resolved as they can be saprophytic, parasitic or shift between both lifestyles and are also classified as primary pathogen able to infect healthy tissue. We argue that Alternaria spp. does not act as primary pathogen, but only as a necrosis-dependent opportunist. We studied the infection biology of Alternaria spp. under controlled conditions and monitored disease prevalence in real orchards and validated our ideas by applying fungicide-free treatments in 3-years field experiments. Alternaria spp. isolates were not able to induce necroses in healthy tissue, but only when prior induced damages existed. Next, leaf-applied fertilizers, without fungicidal effect, reduced Alternaria-associated symptoms (- 72.7%, SE: ± 2.5%) with the same efficacy as fungicides. Finally, low leaf magnesium, sulphur, and manganese concentrations were consistently linked with Alternaria-associated leaf blotch. Fruit spot incidence correlated positively with leaf blotch, was also reduced by fertilizer treatments, and did not expand during storage unlike other fungus-mediated diseases. Our findings suggest that Alternaria spp. may be a consequence of leaf blotch rather than its primary cause, as it appears to colonize the physiologically induced leaf blotch. Taking into account existing observations that Alternaria infection is connected to weakened hosts, the distinction may appear slight, but is of great significance, as we can now (a) explain the mechanism of how different stresses result in colonization with Alternaria spp. and (b) substitute fungicides for a basic leaf fertilizer. Therefore, our findings can result in significant decreases in environmental costs due to reduced fungicide use, especially if the same mechanism applies to other crops.

Project description:As an endoplasmic reticulum (ER) stress sensor, inositol-requiring enzyme 1 (IRE1) splices the bZIP60 mRNA, and produces an active bZIP60 transcription factor that regulates genes involved in the unfolded protein response (UPR) during ER stresses. This IRE1-bZIP60 pathway is conserved in plant species and recently implicated in plant-pathogen interaction. However, it is unclear whether this IRE1-bZIP60 pathway is involved in Nicotiana attenuata resistance to necrotic fungal pathogen, Alternaria alternata. In this study, transcriptional levels of chaperone protein genes, including luminal binding protein (BiP), protein disulfide isomerase (PDI), calnexin 1-like (CNX 1-like), and calreticulin (CRT), and genes involved in IRE1-bZIP60 pathway, were all significantly induced in N. attenuata leaves after A. alternata inoculation. Silencing IRE1 or bZIP60 led to N. attenuata plants more susceptible to A. alternata, which were associated with reduced gene expressions of Feruloyl-CoA 6'-hydroxylase 1 (F6'H1), a gene encoding a key enzyme for phytoalexin scopoletin and scopolin biosynthesis. Further, electromobility shift assays (EMSA) indicated that bZIP60 protein of spliced form could directly bind to the promoter region of F6'H1 in vitro. JA signaling pathway is required for N. attenuata resistance to A. alternata. Interestingly, the fungus-elicited transcriptional levels of BiP, PDI, CNX 1-like, CRT, IRE1, and bZIP60(s) were all significantly decreased in JA-deficient or JA-insensitive plants. Meanwhile, those genes were significantly induced by methyl jasmonate (MeJA) when applied exogenously. However, the transcriptional levels of JA-regulated genes allene oxide synthase (AOS) and lipoxygenease 3 (LOX3) were not affected in plants impaired with IRE1-bZIP60 pathway. Thus, it is concluded that IRE1-bZIP60 pathway is required for N. attenuata resistance to A. alternata, and JA signaling pathway plays an important role in the elicitation of chaperone protein genes and IRE1-bZIP60 pathway.

Dataset Information

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

Publications

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

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