Production of optically pure 2,3-butanediol from <Emphasis Type="Italic">Miscanthus floridulus</Emphasis> hydrolysate using engineered <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strains

2,3-Butanediol (2,3-BD) can be produced by fermentation of natural resources like Miscanthus. Bacillus licheniformis mutants, WX-02ΔbudC and WX-02ΔgldA, were elucidated for the potential to use Miscanthus as a cost-effective biomass to produce optically pure 2,3-BD. Both WX-02ΔbudC and WX-02ΔgldA could efficiently use xylose as well as mixed sugars of glucose and xylose to produce optically pure 2,3-BD. Batch fermentation of M. floridulus hydrolysate could produce 21.6 g/L d-2,3-BD and 23.9 g/L meso-2,3-BD in flask, and 13.8 g/L d-2,3-BD and 13.2 g/L meso-2,3-BD in bioreactor for WX-02ΔbudC and WX-02ΔgldA, respectively. Further fed-batch fermentation of hydrolysate in bioreactor showed both of two strains could produce optically pure 2,3-BD, with 32.2 g/L d-2,3-BD for WX-02ΔbudC and 48.5 g/L meso-2,3-BD for WX-02ΔgldA, respectively. Collectively, WX-02ΔbudC and WX-02ΔgldA can efficiently produce optically pure 2,3-BD with M. floridulus hydrolysate, and these two strains are candidates for industrial production of optical purity of 2,3-BD with M. floridulus hydrolysate.     

Expression Profiles of Pathogenesis-Related Gene, <Emphasis Type="Italic">TaLr35PR1</Emphasis>, as it Relate to <Emphasis Type="Italic">Lr35</Emphasis>-Mediated Adult Plant Leaf Rust Resistance

Plants have developed sophisticated mechanisms to combat pathogen infection. One of the acquired modes in response to pathogen attack is the production of the pathogenesis-related (PR) proteins. Our earlier studies reported that TaLr35PR1, a PR1 gene encoding a protein with conserved serine carboxypeptidase (SCP) domain, has been cloned from wheat near-isogenic line TcLr35. However, the involvement of TaLr35PR1 in wheat growth and Lr35-mediated adult resistance to Puccinia triticina remains unclear. Here, we showed that TaLr35PR1 was strongly induced by P. triticina in wheat plant containing Lr35 (TcLr35), in which the expression level of TaLr35PR1 significantly increased and reached the maximum at 12 hpi. The accumulations of TaLr35PR1 increased stably and showed significant peak challenged by P. triticina at different growth and development periods of TcLr35 wheat while it maintained similar level and changed little in mock inoculated. Western blotting was conducted to confirm that TaLr35PR1 protein was increasingly accumulated in the TcLr35 adult plants after P. triticina inoculation and maintained at a similar level from 120 to 168 h post-inoculation. Similar to the expression patterns of TaLr35PR1 at RNA levels, the accumulations of TaLr35PR1 protein were weak in the seedling stage and then increased to the peak and kept constant levels at the mature stage which is consistent with the expression feature of Lr35 gene as an adult plant resistance gene. All these findings suggest that TaLr35PR1 is involved in wheat growth and Lr35-mediated adult wheat defense response to leaf rust pathogen attack.     

Enhanced production of 2,3-butanediol by engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> through fine-tuning of pyruvate decarboxylase and NADH oxidase activities

Background

2,3-Butanediol (2,3-BD) is a promising compound for various applications in chemical, cosmetic, and agricultural industries. Pyruvate decarboxylase (Pdc)-deficient Saccharomyces cerevisiae is an attractive host strain for producing 2,3-BD because a large amount of pyruvate could be shunted to 2,3-BD production instead of ethanol synthesis. However, 2,3-BD yield, productivity, and titer by engineered yeast were inferior to native bacterial producers because of the following metabolic limitations. First, the Pdc-deficient yeast showed growth defect due to a shortage of C₂-compounds. Second, redox imbalance during the 2,3-BD production led to glycerol formation that lowered the yield.

Results

To overcome these problems, the expression levels of Pdc from a Crabtree-negative yeast were optimized in S. cerevisiae. Specifically, Candida tropicalis PDC1 (CtPDC1) was used to minimize the production of ethanol but maximize cell growth and 2,3-BD productivity. As a result, productivity of the BD5_G1CtPDC1 strain expressing an optimal level of Pdc was 2.3 folds higher than that of the control strain in flask cultivation. Through a fed-batch fermentation, 121.8 g/L 2,3-BD was produced in 80 h. NADH oxidase from Lactococcus lactis (noxE) was additionally expressed in the engineered yeast with an optimal activity of Pdc. The fed-batch fermentation with the optimized 2-stage aeration control led to production of 154.3 g/L 2,3-BD in 78 h. The overall yield of 2,3-BD was 0.404 g 2,3-BD/g glucose which corresponds to 80.7% of theoretical yield.

Conclusions

A massive metabolic shift in the engineered S. cerevisiae (BD5_G1CtPDC1_nox) expressing NADH oxidase was observed, suggesting that redox imbalance was a major bottleneck for efficient production of 2,3-BD by engineered yeast. Maximum 2,3-BD titer in this study was close to the highest among the reported microbial production studies. The results demonstrate that resolving both C₂-compound limitation and redox imbalance is critical to increase 2,3-BD production in the Pdc-deficient S. cerevisiae. Our strategy to express fine-tuned PDC and noxE could be applicable not only to 2,3-BD production, but also other chemical production systems using Pdc-deficient S. cerevisiae.

     

Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain BRC1 with increased inulinase activity

A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L^?1. Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L^?1, showing a high theoretical yield of 92.3%.     

Transgenic Tea Over-expressing <Emphasis Type="Italic">Solanum tuberosum Endo</Emphasis>-1,3-beta-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase Gene Conferred Resistance Against Blister Blight Disease

Tea (Camellia sinensis [L.] O. Kuntze) plant, one of the most important plantation crops in the world, is infected by a fungus called Exobasidium vexans leading to dreaded blister blight disease. The disease may result in crop losses up to 35% which directly affect the tea industry. Solanum tuberosum endo-1,3-beta-d-glucanase was cloned into tea genome via Agrobacterium-mediated transformation. The transformation event initially gave 32 kanamycin-resistant plantlets, out of which PCR analysis confirmed only 10 plantlets about the integration of transgene in the plant genome. Real-time PCR study detected transgene expression in six transgenic plantlets. Upregulation of endogenous C. sinensis pathogenesis-related (PR) genes like PR3 (chitinase I) gene and PR5 (thaumatin-like protein) gene also occurred in transgenic plantlets. Detached leaf infection assay showed resistance to E. vexans in greenhouse-acclimated transgenic plantlets. An inhibitory activity against E. vexans was noticed on the detached leaves of transgenic plantlets compared to control. Transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area unlike the formation of fungal lesion on control plantlet. Thus, it can be inferred that constitutive expression of the potato endo-1,3-beta-d-glucanase gene can be a strategy to produce blister blight-resistant tea.     

Metabolic engineering of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and in silico investigation for enhanced 2,3-butanediol production

Objectives

To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated.

Results

Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta-ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation.

Conclusions

K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity.

     

Engineering cofactor flexibility enhanced 2,3-butanediol production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Enzymatic reduction of acetoin into 2,3-butanediol (2,3-BD) typically requires the reduced nicotinamide adenine dinucleotide (NADH) or its phosphate form (NADPH) as electron donor. Efficiency of 2,3-BD biosynthesis, therefore, is heavily influenced by the enzyme specificity and the cofactor availability which varies dynamically. This work describes the engineering of cofactor flexibility for 2,3-BD production by simultaneous overexpression of an NADH-dependent 2,3-BD dehydrogenase from Klebsiella pneumoniae (KpBudC) and an NADPH-specific 2,3-BD dehydrogenase from Clostridium beijerinckii (CbAdh). Co-expression of KpBudC and CbAdh not only enabled condition versatility for 2,3-BD synthesis via flexible utilization of cofactors, but also improved production stereo-specificity of 2,3-BD without accumulation of acetoin. With optimization of medium and fermentation condition, the co-expression strain produced 92 g/L of 2,3-BD in 56 h with 90% stereo-purity for (R,R)-isoform and 85% of maximum theoretical yield. Incorporating cofactor flexibility into the design principle should benefit production of bio-based chemical involving redox reactions.     

Effects of transgenic expression of <Emphasis Type="Italic">Brevibacterium linens</Emphasis> methionine gamma lyase (MGL) on accumulation of <Emphasis Type="Italic">Tylenchulus semipenetrans and key aminoacid contents</Emphasis> in <Emphasis Type="Italic">Carrizo citrange</Emphasis>

Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the β-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.     

Strains of <Emphasis Type="Italic">Bacillus</Emphasis> ssp. regulate wheat resistance to <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk.

The ability of Bacillus subtilis Cohn and Bacillus thuringiensis Berliner to induce systemic resistance in wheat plants to the casual agent of Septoria nodorum Berk., blotch has been studied. It has been shown that strains of Bacillus ssp. that possess the capacity for endophytic survival have antagonistic activity against this pathogen in vitro. A reduction of the degree of Septoria nodorum blotch development on wheat leaves under the influence of Bacillus spp. was accompanied by the suppression of catalase activity, an increase in peroxidase activity and H₂O₂ content, and expression of defence related genes such us PR-1, PR-6, and PR-9. It has been shown that B. subtilis 26 D induces expression levels of wheat pathogenesis-related (PR) genes which marks a SA-dependent pathway of sustainable development and that B. thuringiensis V-5689 and V-6066 induces a JA/ET-dependent pathway. These results suggest that these strain Bacillus spp. promotes the formation of wheat plant resistance to S. nodorum through systemic activation of the plant defense system. The designed bacterial consortium formed a complex biological response in wheat plants infected phytopathogen.     

Overexpression of soybean <Emphasis Type="Italic">GmERF9</Emphasis> enhances the tolerance to drought and cold in the transgenic tobacco

Ethylene response factors (ERFs) are widespread in plants, which are widely involved in plant response to biotic and abiotic stress. In this research, a soybean gene, GmERF9, was identified and the function was characterized. The results showed that GmERF9 contained a typical AP2/ERF binding domain and a putative nuclear localization signal sequence. The real-time fluorescence quantitative PCR (qPCR) revealed that the expression of GmERF9 could be induced by ethylene (ET), abscisic acid (ABA), drought, salt and cold stresses. GmERF9 protein could specifically bind to the GCC-box and activate the expression of the reporter gene in the yeast cells and tobacco leaves. Overexpression of GmERF9 enhanced the expression of pathogenesis-related (PR) genes, including PR1, PR2, Osmotin (PR5), and SAR8.2. Also, the overexpression of GmERF9 increased the accumulation of proline and soluble carbohydrate, and decreased the accumulation of malondialdehyde under drought and cold stresses in the transgenic tobacco compared to the wild type (WT) tobacco, which indicated that GmERF9 enhanced the tolerance to drought and cold stresses in the transgenic tobacco. In summary, the function of GmERF9 is involved in the response to environmental stresses for plants, which can be used as a candidate gene for genetic engineering of crops.     

Enhanced <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> immune responses caused by heterologous plant genes from <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Background

Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F₁ hybrid was proposed.

Results

By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F₁ hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance.

Conclusions

Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F₁ hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.

     

Strategies for efficient and economical 2,3-butanediol production: new trends in this field

2,3-Butanediol (2,3-BD) is a promising bulk chemical with a potentially wide range of applications e.g., in the manufacture of printing inks, perfumes, synthetic rubber, fumigants, antifreeze agents, fuel additives, foodstuffs and pharmaceuticals. Its high heating value and ability to increase the octane number of fuels make 2,3-BD a promising drop-in fuel. It can also be converted to methyl-ethyl ketone (MEK), which is considered an effective liquid fuel additive. After combination with MEK and hydrogenation reaction, 2,3-BD can be converted to octane, which is used to produce high-quality aviation fuel. Currently 2,3-BD is mainly produced on an industrial scale by chemical methods. However, microbiological production of 2,3-BD offers a less expensive and more environmentally friendly alternative to traditional synthesis. This alcohol is generated from hexoses and pentoses mainly by bacterial strains of the genera Klebsiella, Bacillus, Serratia, and Enterobacter, which can convert waste products (such as glycerol and agricultural residues) and excess biomass (such as wood hydrolysates) to 2,3-BD. Recently, a significant improvement in microbial production has been achieved by the screening of efficient natural microbial strains, the application of alternative cost-effective substrates, and the genetic improvement of microbial producers. Furthermore, Klebsiella strains, which are regarded the most efficient natural 2,3-BD producers, have been subjected to genetic modifications aiming at the removal of pathogenic factors and the development of avirulent strains that could be used for the safe production of the diol. This review summarizes existing knowledge and experience concerning various strategies for efficient and economical microbial production of 2,3-BD.     

<Emphasis Type="Italic">Alternaria brassicae</Emphasis> interactions with the model Brassicaceae member <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> closely resembles those with Mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

Alternaria leaf blight, a disease of oilseed Brassicas is caused by a necrotrophic phytopathogenic fungus Alternaria brassicae. The details of its pathogenesis and defence responses elicited in the host upon infection have not been thoroughly investigated. Here, Arabidopsis accession Gre-0 was identified to be highly susceptible to A. brassicae. A comparative histopathological analysis for disease progression and plant responses to A. brassicae in Arabidopsis and Brassica juncea revealed significant similarities between the two compatible pathosystems. Interestingly, in both the compatible hosts, ROS accumulation, cell death and callose deposition correlated with the development of the disease. Based on our results we propose that Arabidopsis-Alternaria brassicae can be an apt model pathosystem since it emulates the dynamics of the pathogen interaction with its natural host- Brassicas. The existing genetic diversity in Arabidopsis can be a starting point to screen for variation in responses to Alternaria leaf blight. Furthermore, several tools available for Arabidopsis can facilitate the dissection of genetic and molecular basis of resistance.     

Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants

Main conclusion

Resistance against anthracnose fungi was enhanced in transgenic pepper plants that accumulated high levels of a carboxylesterase, PepEST in anthracnose-susceptible fruits, with a concurrent induction of antioxidant enzymes and SA-dependent PR proteins. A pepper esterase gene (PepEST) is highly expressed during the incompatible interaction between ripe fruits of pepper (Capsicum annuum L.) and a hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides). In this study, we found that exogenous application of recombinant PepEST protein on the surface of the unripe pepper fruits led to a potentiated state for disease resistance in the fruits, including generation of hydrogen peroxide and expression of pathogenesis-related (PR) genes that encode mostly small proteins with antimicrobial activity. To elucidate the role of PepEST in plant defense, we further developed transgenic pepper plants overexpressing PepEST under the control of CaMV 35S promoter. Molecular analysis confirmed the establishment of three independent transgenic lines carrying single copy of transgenes. The level of PepEST protein was estimated to be approximately 0.002 % of total soluble protein in transgenic fruits. In response to the anthracnose fungus, the transgenic fruits displayed higher expression of PR genes, PR3, PR5, PR10, and PepThi, than non-transgenic control fruits did. Moreover, immunolocalization results showed concurrent localization of ascorbate peroxidase (APX) and PR3 proteins, along with the PepEST protein, in the infected region of transgenic fruits. Disease rate analysis revealed significantly low occurrence of anthracnose disease in the transgenic fruits, approximately 30 % of that in non-transgenic fruits. Furthermore, the transgenic plants also exhibited resistance against C. acutatum and C. coccodes. Collectively, our results suggest that overexpression of PepEST in pepper confers enhanced resistance against the anthracnose fungi by activating the defense signaling pathways.

     

Cassava <Emphasis Type="Italic">C-repeat binding factor 1</Emphasis> gene responds to low temperature and enhances cold tolerance when overexpressed in Arabidopsis and cassava

Key message

Reactive oxygen species (ROS) oxidize methionine to methionine sulfoxide (MetSO) and thereby inactivate proteins. Methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Our results show that Arabidopsis thaliana MSR enzyme coding gene MSRB8 is required for effector-triggered immunity and containment of stress-induced cell death in Arabidopsis.

Abstract

Plants activate pattern-triggered immunity (PTI), a basal defense, upon recognition of evolutionary conserved molecular patterns present in the pathogens. Pathogens release effector molecules to suppress PTI. Recognition of certain effector molecules activates a strong defense, known as effector-triggered immunity (ETI). ETI induces high-level accumulation of reactive oxygen species (ROS) and hypersensitive response (HR), a rapid programmed death of infected cells. ROS oxidize methionine to methionine sulfoxide (MetSO), rendering several proteins nonfunctional. The methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Though a few plant MSR genes are known to provide tolerance against oxidative stress, their role in plant–pathogen interaction is not known. We report here that activation of cell death by avirulent pathogen or UV treatment induces expression of MSRB7 and MSRB8 genes. The T-DNA insertion mutant of MSRB8 exaggerates HR-associated and UV-induced cell death and accumulates a higher level of ROS than wild-type plants. The negative regulatory role of MSRB8 in HR is further supported by amiRNA and overexpression lines. Mutants and overexpression lines of MSRB8 are susceptible and resistant respectively, compared to the wild-type plants, against avirulent strains of Pseudomonas syringae pv. tomato DC3000 (Pst) carrying AvrRpt2, AvrB, or AvrPphB genes. However, the MSRB8 gene does not influence resistance against virulent Pst or P. syringae pv. maculicola (Psm) pathogens. Our results altogether suggest that MSRB8 function is required for ETI and containment of stress-induced cell death in Arabidopsis.

     

Salicylic acid promotes autophagy via NPR3 and NPR4 in <Emphasis Type="Italic">Arabidopsis</Emphasis> senescence and innate immune response

In Arabidopsis thaliana, the non-expresser pathogenesis-related (NPR) multigene family members NPR1, NPR3, and NPR4 are necessary for salicylic acid (SA) perception. NPR3 and NPR4 are the CUL3 E3-ligase substrate adaptors allowing for the ubiquitination and turnover of NPR1 by the 26s proteasome. Concurrently, roots treated with the SA agonist benzothiadiazole accumulate autophagic bodies via NPR1-dependent signal pathway. However, the mechanisms by which NPR3 and NPR4 regulate autophagy remain unclear. In the present study, using single, double, and triple npr1-, npr3-, and npr4-null mutants and wild-type plants, the following results were obtained: (1) leaf senescence progressed faster in npr3/npr4 mutants than in wild type, suggesting that NPR3 and NPR4 negatively regulated leaf senescence. Moreover, npr3/npr4 promoted the expression of pathogenesis-related 1 (PR1) gene and enhanced resistance in response to avirulent pathogen infections suppressing cell death. Still, all mutants had similar SA levels, suggesting that NPR3 and NPR4 positive regulation of cell death and disease resistance was not associated with SA levels; (2) the number of autophagosomes, ATG7, and ATG8a-phosphatidylethanolamine and the concentration of free green-fluorescence protein were lower in npr3/npr4 mutants than in wild-type plants, indicating that NPR3 and NPR4 affected the two ubiquitination-like conjugation systems during the autophagosome formation and degradation of autophagic bodies.     

Comparative iTRAQ proteomic profiling of susceptible and resistant apple cultivars infected by <Emphasis Type="Italic">Alternaria alternata</Emphasis> apple pathotype

Alternaria blotch, caused by the Alternaria alternata apple pathotype (A. alternata AP), is one of serious pathogen of apples. In order to better understand the molecular mechanisms that underlie the defense responses of apple resistance to Alternaria blotch disease, a comparative proteomic approach was applied to analyze of susceptible and resistant apple cultivars response to A. alternata AP infection using iTRAQ (isobaric tags for relative and absolute quantitation) technique. A total of 4225 proteins were identified, and 1226 proteins were quantified. Of the quantified proteins, 280 and 34 expressed differentially (fold change >1.5) at 72 h post-infection (HPI) in the susceptible (“Starking Delicious”) and the resistant (“Jonathan”) apple cultivars, respectively, compared with mock-inoculated controls. Most of the differentially expressed proteins (DEPs) were associated with host plant resistance to pathogens, including signal transduction, stress and defense, and photosynthesis metabolism. Among these proteins, beta-1,3-glucanase(PR2), thaumatin-like protein (PR5), and lipoxygenase were found in both susceptible and resistant hosts. However, endochitinase and (+)-neomenthol dehydrogenase were only detected in the resistant cultivar and increased in abundance in response to the pathogen attack. To study the role of pathogenesis-related (PR) proteins in the early infection process, their expressions at 6, 18, 36, and 72 HPI were analyzed by western blot. It showed that PR5 were accumulated to a high level at 6 HPI in “Jonathan,” while cannot be detected in “Starking Delicious” until 18 HPI. The above results suggested that endochitinase and (+)-neomenthol dehydrogenase, as well as PR5 which exerts function at early stage, play important roles in apple plant against A. alternata AP infestation.