Early Infection Process Research Articles

Ultrastructure of Hirschmanniella diversa early-stage infection in browning rhizomes of Indian lotus.

Browning rhizome (also known as “browning tuber disease”) in Indian lotus (Nelumbo nucifera) caused by the nematode Hirschmanniella diversa is an emerging agronomic problem. In this study, the authors documented the early infection processes of H. diversa in the apices of young rhizomes of Indian lotus by electron microscopy analysis using an artificial inoculation method. Nematodes were attracted to young rhizome apices, invading them via narrow indentations by 4.5 hr after inoculation. Host cells adjacent to the cavity around the invading nematodes were absent and appeared to have disintegrated during infestation. Following contact with the nematodes, host cell walls and cellular contents became electron-dense and less defined, likely due to digestive enzymes secreted by the invading nematodes. Nematodes invaded to a depth of about 1 mm by 24 hr after inoculation, but did not penetrate the plant any further vertically, similar to the observation of browning in mature rhizomes in the field. The authors propose that the invasion sites of young rhizome apices become blackish-brown blotches as rhizomes mature in the field due to oxidation.

Identification of rice seed infection routes of Fusarium fujikuroi inciting bakanae disease of rice

Bakanae disease caused by Gibberella fujikuroi, Sawada, Wollenworth (teleomorph) (anamorph: Fusarium fujikuroi Nirenberg), is emerging as a serious disease of rice in India. Histopathological study of plant tissue during infection can provide valuable insight for understanding the pathogenesis and the mechanism of resistance. The early infection process of F. fujikuroi in rice was observed at microscopic level using light microscopes and scanning electron microscope (SEM). In seed, the pathogen was observed in the husk, embryo and endosperm with maximum colonization in the husk region. Different infectious structures such as swollen tip hyphae, infectious hyphae, appressorium and infection cushion were observed in F. fujikuroi-rice interactions. The estimates of per cent (%) transmission of inoculum (seed-seed; seedlings-seed and florets-seed) revealed maximum transmission of F. fujikuroi through florets followed by seedlings and seed. The present study established that the infection of rice plants by F. fujikuroi can occur through three routes namely seed, seedlings (soil) and florets (air). The information generated in the present study will be valuable for providing an understanding on the pathogenesis and help to device effective strategies for management of bakanae disease of rice.

Exofacial phospholipids at the plasma membrane: ill-defined targets for early infection processes.

The eukaryotic plasma membrane (PM) consists largely of phospholipids and proteins, and separates the intracellular compartments from the extracellular space. It also serves as a signaling platform for cell-to-cell communication and an interaction platform for the molecular crosstalk between pathogens and their target cells. Much research has been done to elucidate the interactions between pathogens and host membrane proteins. However, little is known about the interactions between pathogens and membrane phospholipids, although reports have described a contribution of phospholipids to cell recognition and/or invasion during early infection by diverse pathogens. Thus, during adhesion to the host cell, the obligate intracellular bacterial pathogens Chlamydia spp., the facultative intracellular pathogen Helicobacter pylori and the facultative aerobic pathogen Vibrio parahaemolyticus, interact with exofacial phospholipids. This review focuses on several prominent instances of pathogen interaction with host-cell phospholipids.

Comprehensive analysis of phenotype, microstructure and global transcriptional profiling to unravel the effect of excess copper on the symbiosis between nitrogen-fixing bacteria and Medicago lupulina

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. However, knowledge of the molecular mechanisms, especially the microstructure and global transcriptional profiling, of the symbiosis process under heavy metal contamination is limited. In this study, a heavy metal-tolerant legume, Medicago lupulina, was treated with different concentrations of copper (Cu). The results showed that the early infection process was inhibited and the nodule ultrastructure was changed under 200 mg kg−1 Cu stress. Most infection threads (ITs) were prevented from entering the nodule cells, and few rhizobia were released into the host cells, in which thickening of the plant cell wall and IT wall was observed, demonstrating that rhizobial invasion was inhibited under Cu stress. RNA-seq analysis indicated that a strong shift in gene expression occurred (3257 differentially expressed genes, DEGs). The most pronounced effect was the upregulation of a set of 71 of 73 DEGs for nodule-specific cysteine-rich peptides, which have been shown to control the terminal differentiation of rhizobia in the nodules and to have antimicrobial activity. Various genes for metal transport, chelation binding and antioxidant defence were regulated. In particular, the DEGs for Cu trafficking and detoxification were induced during nodule formation. The DEGs for ethylene (ET) biosynthesis and signalling were also differentially expressed during nodulation, suggesting that the inhibition of nodulation by Cu occurred partially through ET signalling. Furthermore, the genes related to the cell wall were mostly upregulated and most likely involved in cell wall thickening. These findings provide an integrated understanding of the effects of Cu on legume nodule symbiosis at the molecular and phenotypic levels.

Development of leafhopper cell culture to trace the early infection process of a nucleorhabdovirus, rice yellow stunt virus, in insect vector cells

BackgroundIn China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear.MethodsIn present study, we used continuous leafhopper cell culture, immunocytochemical techniques, and transmission electron microscopy to investigate the subcellular distributions of N and P during RYSV infection. Both GST pull-down assay and yeast two-hybrid assay were used to assess the in vitro interaction of N and P. The dsRNA interference assay was performed to study the functional roles of N and P in the assembly of RYSV viroplasm.ResultsHere we demonstrated that N and P colocalized in the nucleus of RYSV-infected Nephotettix cincticeps cell and formed viroplasm-like structures (VpLSs). The transiently expressed N and P are sufficient to form VpLSs in the Sf9 cells. In addition, the interactions of N/P, N/N and P/P were confirmed in vitro. More interestingly, the accumulation of RYSV was significantly reduced when the transcription of N gene or P gene was knocked down by dsRNA treatment.ConclusionsIn summary, our results suggest that N and P are the main viral factors responsible for the formation of viroplasm in RYSV-infected insect cells. Early during RYSV infection in the insect vector, N and P interacted with each other in the nucleus to form viroplasm-like structures, which are essential for the infection of RYSV.

Discovery of selective dengue virus inhibitors using combination of molecular fingerprint-based virtual screening protocols, structure-based pharmacophore model development, molecular dynamics simulations and in vitro studies

Dengue virus is a major issue of tropical and sub-tropical regions. The proliferation of virus results in immense number of deaths each year because of unavailability of on-shelf drugs. This issue necessitates the design of novel anti-Dengue drugs. The protease enzyme pathway is the critical target for drug design due to its significance in the replication, survival and other cellular activities of Dengue virus. Keeping in mind the worsening situation regarding Dengue virus, approximately eighteen million drug-like compounds from the ZINC small molecule database have been screened against Nonstructural Protein 3 (NS3) previously by our group. In this study, in order to investigate the effect of extended time of molecular dynamics (MD) simulations on structural and dynamical profiles of used complexes, simulation run time is increased from 50-ns to 100-ns for the each system. In addition, a well-known Dengue virus inhibitor (MB21) from literature is used as reference structure (positive control) to compare the proposed molecules. Post-processing MD analyses including Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations were conducted to predict binding free energies of inhibitors from derived trajectory frames of MD simulations. Identified compounds are further directed to Quantum-Polarized Ligand Docking (QPLD), molecular fingerprint-based virtual screening of another small molecule database (Otava Drug Like small molecule database), and Structure-based Pharmacophore Modeling (E-Pharmacophore). Finally, cell proliferation and cytotoxicity tests as well as pre- and post-treatment on HUH7 cells infected with DENV2 NGC strain are applied for four identified hit molecules (ZINC36681949, ZINC44921800, ZINC95518765 and ZINC39500661) to check whether these drugs inhibit DENV2 from entry and/or exit pathways. Based on cell-based Dengue quantification assays, there is no effect seen on pre-treatment of cells with these compounds indicating that the early infection processes of virus is not affected. In contrast, the post-treatment of cells with these compounds after Dengue virus infection has resulted in a significant 1 log PFU/ml reduction of the virus infectious titre.

Factors affecting Neofuscicoccum ribis infection and disease progression in blueberry

Botryosphaeria stem blight is an economically important disease of blueberry worldwide. In this study, factors affecting inoculum production, infection and disease progression of Neofusicoccum spp. in blueberries were investigated. Under laboratory conditions conidia of the main three Neofusicoccum species (N. australe, N. parvum and N. ribis) were released from pycnidia at 15–30 °C and under relative humidities (RHs) of 80–100%, with greatest numbers released by N. parvum. The greatest numbers of oozing pycnidia and conidial release occurred at higher temperatures (25–30 °C) and RHs (92–100%). Inoculation of green shoots with different N. parvum and N. ribis conidial concentrations (50 μL of 5 × 104−5 × 106 conidia/ mL) caused 100% incidence but lesion lengths increased with increasing concentrations. Wound age affected N. ribis lesion development, with lesions only observed for 0–7-day-old wounds in soft green shoots and 0–4-day-old wounds for both hard green shoots and trunks. Colonisation length decreased with increasing wound age. Lesions developed on wounded shoots when plants were exposed to 20 or 25 °C and 90 or 100% RH during the early infection processes; and in non-wounded shoots spot-like lesions were observed although N. ribis colonised the stem tissue. Seasons (summer, autumn and winter) had no effect on susceptibility of wounded plants to N. ribis. External lesions only developed in summer-inoculated plants and colonisation length was lower in winter-inoculated plants. Information on host and environmental factors that affect disease development determined by the study will be used to inform the development of control strategies.

Publisher Note

Dengue virus is a major issue of tropical and sub-tropical regions. The proliferation of virus results in immense number of deaths each year because of unavailability of on-shelf drugs. This issue necessitates the design of novel anti-Dengue drugs. The protease enzyme pathway is the critical target for drug design due to its significance in the replication, survival and other cellular activities of Dengue virus. Keeping in mind the worsening situation regarding Dengue virus, approximately eighteen million drug-like compounds from the ZINC small molecule database have been screened against Nonstructural Protein 3 (NS3) previously by our group. In this study, in order to investigate the effect of extended time of molecular dynamics (MD) simulations on structural and dynamical profiles of used complexes, simulation run time is increased from 50-ns to 100-ns for the each system. In addition, a well-known Dengue virus inhibitor (MB21) from literature is used as reference structure (positive control) to compare the proposed molecules. Post-processing MD analyses including Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations were conducted to predict binding free energies of inhibitors from derived trajectory frames of MD simulations. Identified compounds are further directed to Quantum-Polarized Ligand Docking (QPLD), molecular fingerprint-based virtual screening of another small molecule database (Otava Drug Like small molecule database), and Structure-based Pharmacophore Modeling (E-Pharmacophore). Finally, cell proliferation and cytotoxicity tests as well as pre- and post-treatment on HUH7 cells infected with DENV2 NGC strain are applied for four identified hit molecules (ZINC36681949, ZINC44921800, ZINC95518765 and ZINC39500661) to check whether these drugs inhibit DENV2 from entry and/or exit pathways. Based on cell-based Dengue quantification assays, there is no effect seen on pre-treatment of cells with these compounds indicating that the early infection processes of virus is not affected. In contrast, the post-treatment of cells with these compounds after Dengue virus infection has resulted in a significant 1logPFU/ml reduction of the virus infectious titre.

Response of sea fans to infection with Aspergillus sp. (Fungi)

Mass mortalities of sea fans (Gorgonia spp.) off the South and Central American coasts were reported in the 1980s. In some cases, populations never recovered. Widespread tissue mortality in G. ventalina and G. flabellum throughout the Caribbean was reported in 1997. Although colony mortality was apparently lower in the more recent epizootic, the geographic distribution of the disease appeared much greater. Tissue samples of affected colonies were found to be infected with a fungus (Aspergillus sp.) with was shown to be able to cause the disease in healthy sea fans. We describe observations on the early infection process and subsequent development of the disease. Based on these observations, we report the response of Gorgina spp. to infection by Aspergillus.

Transcriptomic analysis of two Beauveria bassiana strains grown on cuticle extracts of the silkworm uncovers their different metabolic response at early infection stage

Beauveria bassiana is an important entomopathogenic fungus which not only widely distributes in the environment but also shows phenotypic diversity. However, the mechanism of pathogenic differences among natural B. bassiana strains has not been revealed at transcriptome-wide level. In the present study, in order to explore the mechanism, two B. bassiana strains with different pathogenicity were isolated from silkworms (Bombyx mori L.) and selected to analyze the gene expression of early stage by culturing on cuticle extracts of the silkworm and using RNA-sequencing technique. A total of 2108 up-regulated and 1115 down-regulated genes were identified in B. bassiana strain GXsk1011 (hyper-virulent strain) compared with B. bassiana strain GXtr1009 (hypo-virulent strain), respectively. The function categorization of differential expressed genes (DEGs) showed that most of them involved in metabolic process, biosynthesis of secondary metabolites, catalytic activity, and some involved in nutrition uptake, adhesion and host defense were also noted. Based on our data, distinct pathogenicity among different strains of B. bassiana may largely attribute to unique gene expression pattern which differed at very early infection process. Most of the genes involved in conidia adhesion, cuticle degradation and fungal growth were up-regulated in hyper-virulent B. bassiana strain GXsk1011. Furthermore, in combination with fungal growth analysis, our research provided a clue that fungal growth may also play an important role during early infection process. The results will help to explain why different B. bassiana strains show distinct pathogenicity on the same host even under same condition. Moreover, the transcriptome data were also useful for screening potential virulence factors.

Comparative iTRAQ proteomic profiling of susceptible and resistant apple cultivars infected by Alternaria alternata 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.

In-Silico Identification of Novel Resistant Genes for Fungal Pathogen Fusarium oxysporum f. sp. cubense Race 4: Causative Agent of Banana Vascular Wilt Disease

Cavendish, the most widely grown banana cultivar, is relatively resistant to Race 1 of Fusarium oxysporum f. sp. cubense (Foc1) that caused widespread Panama disease during the first half of the 20th century but is susceptible to Tropical Race 4 of the Foc (Foc TR4), a threat to world banana production. Foc TR4 can spread into the vascular system of banana roots during the early infection process. The genome of the diploid species Musa acuminata; the ancestor of majority of the triploid banana cultivars has recently been sequenced. The identification for the resistant gene in this cultivar is a challenge for the researchers as the resistant gene against Foc4 has not been identified, the major focus of this research work was to identify the pathogenic genes and some resistant genes against those pathogenic genes, to get some better results for the laboratory work. The four resistant genes I, I-1, I-2, and I-3 were identified which provides resistance against Foc4 in banana. About three pathogenic genes SIX1 (SIX1A, SIX1B, SIX1C), SIX2, and SIX8 of Foc4 were identified. Out of these pathogenic genes SIX8 showed the greatest number of interactions with the resistant proteins. So it is proposed that I-1, I-2 and I-3 genes can be used in the further research work for providing better improvements of the resistive cultivars in banana.

Flagellar motility is necessary for Aeromonas hydrophila adhesion

Adhesion to host surface or cells is the initial step in bacterial pathogenesis, and the adhesion mechanisms of the fish pathogenic bacteria Aeromonas hydrophila were investigated in this study. First, a mutagenesis library of A. hydrophila that contained 332 random insertion mutants was constructed via mini-Tn10 Km mutagenesis. Four mutants displayed the most attenuated adhesion. Sequence analysis revealed that the mini-Tn10 insertion sites in the four mutant strains were flgC(GenBank accession numbers KX261880), cytb4(GenBank accession numbers JN133621), rbsR(GenBank accession numbers KX261881) and flgE(GenBank accession numbers JQ974982). To further study the roles of flgC and flgE in the adhesion of A. hydrophila, some biological characteristics of the wild-type strain B11, the mutants M121 and M240, and the complemented strains C121 and C240 were investigated. The results showed that the mutation in flgC or flgE led to the flagellar motility of A. hydrophila significant reduction or abolishment. flgC was not necessary for flagellar biosynthesis but was necessary for the full motility of A. hydrophila, flgE was involved in both flagellar biosynthesis and motility. The flagellar motility is necessary for A. hydrophila to adhere to the host mucus, which suggests flagellar motility plays crucial roles in the early infection process of this bacterium.

Predictive factors for the appearance of myringosclerosis after myringotomy with ventilation tube placement: randomized study.

Myringotomy with the insertion of ventilation tubes is the most frequent surgical procedure performed in children, and the appearance of myringosclerosis is one of its most frequent long-term complications. The objective of this study is to identify clinical factors and technique variations that may have a relation with the appearance of myringosclerosis, after tube insertion. Patients submitted to myringotomy with transtympanic short-term tube insertion were studied in a longitudinal prospective and analytical cohort study with the prospective randomized open, blinded endpoint (PROBE) methodology, to study the influence of the location of myringotomy (anterior-inferior quadrant or posterior-inferior), directions of the incision (radial or non-radial) and aspiration or not of the middle ear. Our study included 156 patients (297 ears). Myringosclerosis was observed in 35.7% of the operated ears. It appeared more often in patients with greater number of otitis (p=.001) and with greater number of otorrhea episodes (p=.029) and in patients in whom the tympanogram after the tube extraction was type A (according to Jerger´s classification) (p=0.016). We identified myringosclerosis in less patients, if the tube was in the tympanic membrane for less than 12months (p=.009). Myringosclerosis was present more extensively if the tympanic incision was located in the anterior-inferior quadrant, with tympanic involvement superior to 25% (p=.015). The results observed prove that, underlying the appearance of myringosclerosis, there exists an early inflammatory or infectious process and a final cicatricial process. It was also found that when myringotomy is made in the anterior-inferior quadrant, myringosclerosis appears in a higher percentage of the tympanic membrane; therefore, it is not recommended to do the incision in this quadrant, because it may lead to a reduction of the tympanic membrane vibration.

Myeloperoxidase-responsive materials for infection detection based on immobilized aminomethoxyphenol.

There is a strong need for simple and fast diagnostic tools for the detection of wound infection. Immune system-derived enzymes like myeloperoxidase are efficient biomarkers for wound infection that emerge in the early stage infection process. In this study, 5-amino-2-methoxyphenol was functionalized with alkoxysilane to allow visual detection of MPO on carrier materials, for example, in test strips. Indeed, MPO activity was visually detectable in short time in wound background. Oxidation of the substrate was followed spectrophotometrically and proved via HPLC. LC-ESI TOF and NMR analyses unveiled the reaction mechanism and a dimeric reaction product responsible for the visualization of MPO activity. The substrate specificity and sensitivity toward MPO detection was proved and tests with infected wound fluids were successfully performed. The study demonstrates the suitability of the novel MPO substrate for the detection of wound infection and the covalent immobilization on diagnostic carrier materials. Biotechnol. Bioeng. 2016;113: 2553-2560. © 2016 Wiley Periodicals, Inc.

Early Generation of New PrPSc on Blood Vessels after Brain Microinjection of Scrapie in Mice.

ABSTRACTAggregation of misfolded host proteins in the central nervous system is believed to be important in the pathogenic process in several neurodegenerative diseases of humans, including prion diseases, Alzheimer’s disease, and Parkinson’s disease. In these diseases, protein misfolding and aggregation appear to expand through a process of seeded polymerization. Prion diseases occur in both humans and animals and are experimentally transmissible orally or by injection, thus providing a controllable model of other neurodegenerative protein misfolding diseases. In rodents and ruminants, prion disease has a slow course, lasting months to years. Although prion infectivity has been detected in brain tissue at 3 to 4 weeks postinfection (p.i.), the details of early prion replication in the brain are not well understood. Here we studied the localization and quantitation of PrPSc generation in vivo starting at 30 min postmicroinjection of scrapie into the brain. In C57BL mice at 3 days p.i., generation of new PrPSc was detected by immunohistochemistry and immunoblot assays, and at 7 days p.i., new generation was confirmed by real-time quaking-induced conversion assay. The main site of new PrPSc generation was near the outer basement membrane of small and medium blood vessels. The finding and localization of replication at this site so early after injection have not been reported previously. This predominantly perivascular location suggested that structural components of the blood vessel basement membrane or perivascular astrocytes might act as cofactors in the initial generation of PrPSc. The location of PrPSc replication at the basement membrane also implies a role for the brain interstitial fluid drainage in the early infection process.

Tightly regulated expression of Autographa californica multicapsid nucleopolyhedrovirus immediate early genes emerges from their interactions and possible collective behaviors.

To infect their hosts, DNA viruses must successfully initiate the expression of viral genes that control subsequent viral gene expression and manipulate the host environment. Viral genes that are immediately expressed upon infection play critical roles in the early infection process. In this study, we investigated the expression and regulation of five canonical regulatory immediate-early (IE) genes of Autographa californica multicapsid nucleopolyhedrovirus: ie0, ie1, ie2, me53, and pe38. A systematic transient gene-expression analysis revealed that these IE genes are generally transactivators, suggesting the existence of a highly interactive regulatory network. A genetic analysis using gene knockout viruses demonstrated that the expression of these IE genes was tolerant to the single deletions of activator IE genes in the early stage of infection. A network graph analysis on the regulatory relationships observed in the transient expression analysis suggested that the robustness of IE gene expression is due to the organization of the IE gene regulatory network and how each IE gene is activated. However, some regulatory relationships detected by the genetic analysis were contradictory to those observed in the transient expression analysis, especially for IE0-mediated regulation. Statistical modeling, combined with genetic analysis using knockout alleles for ie0 and ie1, showed that the repressor function of ie0 was due to the interaction between ie0 and ie1, not ie0 itself. Taken together, these systematic approaches provided insight into the topology and nature of the IE gene regulatory network.

An S-(hydroxymethyl)glutathione dehydrogenase is involved in conidiation and full virulence in the rice blast fungus Magnaporthe oryzae.

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice blast disease. A compatible interaction requires overcoming plant defense responses to initiate colonization during the early infection process. Nitric oxide (NO) plays important roles in defense responses during host-pathogen interactions. Microbes generally protect themselves against NO-induced damage by using enzymes. Here, we characterized an S-(hydroxymethyl)- glutathione dehydrogenase gene in M. oryzae, MoSFA1, the homologs of which are involved in NO metabolism by specifically catalyzing the reduction of S-nitrosoglutathione (GSNO) in yeasts and plants. As expected from the activities of S-(hydroxymethyl)glutathione dehydrogenase in formaldehyde detoxification and GSNO reduction, MoSFA1 deletion mutants were lethal in formaldehyde containing medium, sensitive to exogenous NO and exhibited a higher level of S-nitrosothiols (SNOs) than that of the wild type. Notably, the mutants showed severe reduction of conidiation and appressoria turgor pressure, as well as significantly attenuated the virulence on rice cultivar CO-39. However, the virulence of MoSFA1 deletion mutants on wounded rice leaf was not affected. An infection assay on barley leaf further revealed that MoSFA1 deletion mutants exhibited a lower infection rate, and growth of infectious hyphae of the mutants was retarded not only in primary infected cells but also in expansion from cell to cell. Furthermore, barley leaf cell infected by MoSFA1 deletion mutants exhibited a stronger accumulation of H2O2 at 24 and 36 hpi. MoSFA1 deletion mutants displayed hypersensitivity to different oxidants, reduced activities of superoxide dismutases and peroxidases, and lower glutathione content in cells, compared with the wild type. These results imply that MoSFA1-mediated NO metabolism is important in redox homeostasis in response to development and host infection of M. oryzae. Taken together, this work identifies that MoSFA1 is required for conidiation and contributes to virulence in the penetration and biotrophic phases in M. oryzae.

Differential expression analysis of a select list of genes in susceptible and resistant heirloom tomatoes with respect to Pseudomonas syringae pv. tomato

Bacterial speck disease of tomato, caused by Pseudomonas syringae pv. tomato (Pst) is one of the most devastating diseases of tomato in the world. To investigate plant responses activated during plant-pathogen interaction, we studied the expression analysis of selected defense-response genes and the microbe or pathogen associated molecular patterns (MAMP or PAMP) triggered immunity (PTI) marker genes in heirloom tomatoes challenged with virulent Pst DC3000. Transcript levels of the defense response genes, including SlPR1a (pathogenesis related proteins), SlPR-Q′b (β-1,3-glucanase), SlGST (Glutathione S-transferase) and peroxidase were up-regulated in the resistant cultivars Orange Strawberry and Amishpaste. The PTI marker genes SlPTI5 (encode a pathogen-inducible ethylene response element-binding protein-like transcription factor) and SlFLS2 (a leucine rich-repeat receptor like kinase) were strongly up-regulated in the incompatible reaction to resistant cultivar Orange Strawberry. On the other hand, transcripts from all tested genes were down-regulated in the compatible interaction in susceptible cultivars Yellow Pear and Brandywine. The induction of defense response and PTI marker genes occurred in the early infection process at 3 days post-inoculation (dpi) and consistent with lower levels of disease severity in resistant cultivars was observed than in susceptible cultivars. Our results contribute to the body of information on bacterial speck disease resistance in an economically important crop tomato.

Replication of Chinese sacbrood virus in primary cell cultures of Asian honeybee (Apis cerana).

A primary cell culture system was established for the first time from embryonic tissues of Asian honeybee, Apis cerana, and used to trace the early infection process of Chinese sacbrood virus (CSBV), an iflavirus in the family Iflaviridae. A monolayer of epithelium-like cells of A. cerana, approximately 8-10 μm in diameter, was grown in Kimura's insect medium at 28 °C within 3-4 days of setting up the cultures. Such cultured cells were inoculated with CSBV purified from infected larvae or pupae for 2 h. In electron and confocal micrographs, viral particles accumulated as filamentous or vesicular inclusions in the cytoplasm of infected cultured cells at 36 h post-inoculation (hpi). Real-time quantitative RT-PCR assay showed that the expression levels of four cistrons of CSBV in the cultured cells increased rapidly between 12 and 48 hpi. This newly established primary cell culture derived from A. cerana will be useful for further studies of infection caused by CSBV.