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Plant antimicrobial peptides (AMPs) as a part of plant defense responses, are small soluble defense molecules which can inhibit the growth of pathogens. This study evaluates the effect of an antimicrobial peptide obtained from Allium sativum (AsR416) on Rhizoctonia solani (AG1-IA) the causal agent of rice sheath blight, in vitro and in vivo conditions. Firstly, the obtained results revealed that AsR416 (100 mg ml−1) inhibited the growth and sclerotia production of R. solani AG1-IA. Furthermore, these results showed the mode of action and mechanisms of AsR416 effect in inhibiting sclerotia formation of R. solani AG1-IA via metabolomics tools. AsR416 decreased the biomass of R. solani AG1-IA in liquid culture. In addition, nitro blue tetrazolium and evans blue staining methods revealed that the antimicrobial peptide induced O2− formation in the hyphal cells and mycelia cell death of R. solani AG1-IA, respectively. AsR416 delayed the pathogen infection process and decreased the severity of rice sheath blight disease in vitro and in vivo conditions. AsR416 reduced activity of cellulase, which is one of the virulence factors of this pathogen. The number of sclerotia decreased on plants treated with AsR416 after 2 months compared with the control. Considering the need to reduce application of hazardous synthetic fungicides against pathogenic fungi, using AMPs could be a successful method to increase rice production and reduce the use of chemical fungicides against sheath blight disease.

Background SA (Salicylic acid) and JA (Jasmonic acid)/ET (Ethylene) are the defense and growth hormone regulators involved in alleviating the biotic and abiotic stresses. WRKY and NAC genes are contributors to plant resistance due to their active role in SA and JA/ET defense mechanism. Methods WRKY and NAC transcription factors (ptHR293 and ptHR759, respectively) were serially selected (by performing cDNA library functional screening, homology analysis, antioxidant enzymes, ROS burst, callose deposition and qRT-PCR analysis) from Pinellia ternata and transformed into Nicotiana benthamiana. Hybrids were generated to analyze the stability of disease resistance. UPLC-QTOF-MS was performed to study the bioactive compounds. Results Study demonstrated that ptHR293 and ptHR759 had potential to trigger ROS burst and callose deposition in N. benthamiana together with the activation of PR-genes and antioxidant enzymes. In transformed N. benthamiana, relative expression of PR-1a (Pathogenesis related-1a) and PDF1.2 (Plant defensin 1.2) was upregulated upto 21 folds and 4 folds for ptHR293 transformed N. benthamiana. While PR-1a and PDF1.2 exhibited 8 folds and 26 folds upregulation for ptHR759 transformed N. benthamiana. ptHR293 + ptHR759-F1 hybrids also exhibited a significant level of PR-gene expression. Significantly high resistance against Botrytis cinerea without influencing the standard seed germination, root and shoot length of transformed N. benthamiana was also observed. A significant induction of bioactive compounds was also observed in ptHR293 transformed N. benthamiana. Conclusion Conclusively, heterologous transcription factors, ptHR293 and ptHR759, perform their specific role in the activation of SA and JA/ET mediated defense mechanism.

Even in a natural ecosystem, plants are continuously threatened by various microbial diseases. To save themselves from these diverse infections, plants build a robust, multilayered immune system through their natural chemical compounds. Among the several crucial bioactive compounds possessed by plants’ immune systems, antimicrobial peptides (AMPs) rank in the first tier. These AMPs are environmentally friendly, anti-pathogenic, and do not bring harm to humans. Antimicrobial peptides can be isolated in several ways, but recombinant protein production has become increasingly popular in recent years, with the Escherichia coli expression system being the most widely used. However, the efficacy of this expression system is compromised due to the difficulty of removing endotoxin from its system. Therefore, this review suggests a high-throughput cDNA library-based plant-derived AMP isolation technique using the Bacillus subtilis expression system. This method can be performed for large-scale screening of plant sources to classify unique or homologous AMPs for the agronomic and applied field of plant studies. Furthermore, this review also focuses on the efficacy of plant AMPs, which are dependent on their numerous modes of action and exceptional structural stability to function against a wide range of invaders. To conclude, the findings from this study will be useful in investigating how novel AMPs are distributed among plants and provide detailed guidelines for an effective screening strategy of AMPs.

Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms. We chose Bacillus subtilis as a target pathogen indicator and transferred the Aegilops tauschii Cosson cDNA library into B. subtilis cells. Expression of the candidate antimicrobial gene can inhibit B. subtilis cell growth. Using this strategy, we screened six genes that have an internal effect on the indicator bacteria. Then, the secreted proteins were extracted and tested; two genes, AtR100 and AtR472, were found to have strong external antimicrobial activities with broad-spectrum resistance against Xanthomonas oryzae pv. oryzicola, Clavibacter fangii, and Botrytis cinerea. Additionally, thermal stability tests indicated that the antimicrobial activities of both proteins were thermostable. Furthermore, these two proteins exhibited no significant hemolytic activities. To test the feasibility of application at the industrial level, liquid fermentation and spray drying of these two proteins were conducted. Powder dilutions were shown to have significant inhibitory effects on B. cinerea. Fluorescence microscopy and flow cytometry results showed that the purified protein impaired and targeted the cell membranes. This study revealed that these two antimicrobial peptides could potentially be used for replacing antibiotics, which would provide the chance to reduce the emergence of drug resistance.

Endophytic microbial-communities have specific beneficial functions and are considered key drivers for host plant health. The removing-PCR (R-PCR) is a simple culture-independent cost-effective method to identify endophytic microbial-communities. Microbial communities from maize plant grown in different soil types were identified and characterized via the R-PCR and 16S rRNA sequencing. Culture-dependent microbial community identified through 16S rRNA gene sequencing, further these bacterial communities screened for antagonistic assay against Rhizoctonia solani WH1, in vitro compatibility tests, plant-growth-promoting traits and BIOLOG identification. After that, synthetic-communities (SycomA and SycomB) were prepared by mixing different compatible bacterial-strains to use as an inoculant to suppress pathogens of maize. We identified 167 bacterial operational taxonomic units (OTUs) and unexpected 8 fungal OTUs through the R-PCR, whereas, 95 bacterial OTUs via 16S rRNA sequencing from maize leaves and roots. SycomA and SycomB treatments suppressed the disease level and promoted growth attributes more effectively as compare to the single bacterial-strain and control treatments. This study establishes an efficient approach to isolate, identify and characterize diverse endophytic microbial-community assembly in maize leaves and roots, to successfully apply particular microbes to improve crop growth in soils affected by soil-borne-pathogens.

Key message Nine transgenes from different categories, viz. plant defense response genes and anti-apoptosis genes, played combined roles in maize to inhibit the necrotrophic pathogens Rhizoctonia solani and Bipolaris maydis. Maize sheath blight and southern corn leaf blight are major global threats to maize production. The management of these necrotrophic pathogens has encountered limited success due to the characteristics of their lifestyle. Here, we presented a transgenic pyramiding breeding strategy to achieve nine different resistance genes integrated in one transgenic maize line to combat different aspects of necrotrophic pathogens. These nine genes, selected from two different categories, plant defense response genes (Chi, Glu, Ace-AMP1, Tlp, Rs-AFP2, ZmPROPEP1 and Pti4), and anti-apoptosis genes (Iap and p35), were successfully transferred into maize and further implicated in resistance against the necrotrophic pathogens Rhizoctonia solani and Bipolaris maydis. Furthermore, the transgenic maize line 910, with high expression levels of the nine integrated genes, was selected from 49 lines. Under greenhouse and field trial conditions, line 910 showed significant resistance against maize sheath blight and southern corn leaf blight diseases. Higher-level resistance was obtained after the pyramiding of more resistance transgenes from different categories that function via different mechanisms. The present study provides a successful strategy for the management of necrotrophic pathogens.

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. Electronic supplementary material The online version of this article (10.1186/s12870-018-1598-5) contains supplementary material, which is available to authorized users.

Antimicrobial genes are distributed in all forms of life and provide a primary defensive shield due to their unique broad-spectrum resistance activities. To better isolate these genes, we used the Bacillus subtilis expression system as the host cells to build Oryza rufipogon Griff cDNA libraries and screen potential candidate genes from the library at higher flux using built-in indicator bacteria. We observed that the antimicrobial peptides OrR214 and OrR935 have strong antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria, as well as several fungal pathogens. Owing to their high thermal and enzymatic stabilities, these two peptides can also be used as field biocontrol agents. Furthermore, we also found that the peptide OrR214 (MIC 7.7&ndash, 10.7 &mu, M) can strongly inhibit bacterial growth compared to polymyxin B (MIC 5&ndash, 25 &mu, M) and OrR935 (MIC 33&ndash, 44 &mu, M). The cell flow analysis, reactive oxygen burst, and electron microscopy (scanning and transmission electron microscopy) observations showed that the cell membranes were targeted by peptides OrR214 and OrR935, which revealed the mode of action of bacteriostasis. Moreover, the hemolytic activity, toxicity, and salt sensitivity experiments demonstrated that these two peptides might have the potential to be used for clinical applications. Overall, OrR214 and OrR935 antimicrobial peptides have a high-throughput bacteriostatic activity that acts as a new form of antimicrobial agent and can be used as a raw material in the field of drug development.

Yeast one- (Y1H) and two-hybrid (Y2H) assays are widely used to study transcription factors (TFs) and protein-protein interactions (PPIs), respectively. Here we combined the Y1H and Y2H into a yeast library-hybrid (YLH) assay, which can systematically screen transcription factors (TFs) and PPIs in one experimental pipeline. In typical TFs, the DNA-binding domain (DBD) and activation domain (AD) evolved separately, but were covalently linked. In the YLH assay, TFs are identified based on functionally conserved ADs, whereas in various Y1H assays TFs are identified based on DBDs. Using the YLH method, we isolated 51 pairs of maize-Rhizoctonia PPIs and 38 novel Rhizoctonia solani TFs. TFs and PPIs related to pathogen virulence and plant resistance responses were isolated by the YLH assay. Our results show that 57.75% of isolated TFs contain typical DBDs such as Zn2Cys6, nucleic acid-binding OB-fold, winged helix repressor DNA-binding, and zinc finger CCHC-type. Key PPI pairs related to major functional categories such as metabolism and cellular signalling were obtained. The percentage of verified PPIs is 69.39%. We proved that common TFs have nonspecific or broad-spectrum activities in the yeast plasmid gene expression system. YLH screening can be conducted on library scales to systematically reveal possible TFs and PPIs at the same time.

Antimicrobial genes are found in all classes of life. To efficiently isolate these genes, we used Bacillus subtilis and Escherichia coli as target indicator bacteria and transformed them with cDNA libraries. Among thousands of expressed proteins, candidate proteins played antimicrobial roles from the inside of the indicator bacteria (internal effect), contributing to the sensitivity (much more sensitivity than the external effect from antimicrobial proteins working from outside of the cells) and the high throughput ability of screening. We found that B. subtilis is more efficient and reliable than E. coli. Using the B. subtilis expression system, we identified 19 novel, broad-spectrum antimicrobial genes. Proteins expressed by these genes were extracted and tested, exhibiting strong external antibacterial, antifungal and nematicidal activities. Furthermore, these newly isolated proteins could control plant diseases. Application of these proteins secreted by engineered B. subtilis in soil could inhibit the growth of pathogenic bacteria. These proteins are thermally stable and suitable for clinical medicine, as they exhibited no haemolytic activity. Based on our findings, we speculated that plant, animal and human pathogenic bacteria, fungi or even cancer cells might be taken as the indicator target cells for screening specific resistance genes.

The situation of drug resistance has become more complicated due to the scarcity of plant resistance genes, and overcoming this challenge is imperative. Isatis indigotica has been used for the treatment of wounds, viral infections, and inflammation for centuries. Antimicrobial peptides (AMPs) are found in all classes of life ranging from prokaryotes to eukaryotes. To identify AMPs, I. indigotica was explored using a novel, sensitive, and high-throughput Bacillus subtilis screening system. We found that IiR515 and IiR915 exhibited significant antimicrobial activities against a variety of bacterial (Xanthomonas oryzae, Ralstonia solanacearum, Clavibacter michiganensis, and C. fangii) and fungal (Phytophthora capsici and Botrytis cinerea) pathogens. Scanning electron microscope and cytometric analysis revealed the possible mechanism of these peptides, which was to target and disrupt the bacterial cell membrane. This model was also supported by membrane fluidity and electrical potential analyses. Hemolytic activity assays revealed that these peptides may act as a potential source for clinical medicine development. In conclusion, the plant-derived novel AMPs IiR515 and IiR915 are effective biocontrol agents and can be used as raw materials in the drug discovery field.

Table S1. Primers used for qRT-PCR. Figure S1. Agarose gel of cDNA inserts. M, 100â bp marker. Figure S2. Hypersensitive response induced by ptHR genes on L. esculentum, G. hirustum, N. benthamiana and P. ternata leaves. Table S2. NCBI blast results showing homology with known sequences. Table S3. NCBI blast results showing homology with transcription factors. Table S4. NCBI blast results showing no homology with known sequences. Figure S3. Relative expression levels of pathogenesis-related genes in N. benthamiana. Figure S4. Relative expression levels of ptHR941 and ptHR375 genes in transformed N. benthamiana. Figure S5. Mass spectra for the induced bioactive compounds detected in ptHR375 transformed N. benthamiana. Table S5. List of induced bioactive compounds present in ptHR375 transformed N. benthamiana. (PDF 1033 kb)

Plant growth-promoting bacteria (PGPB) may trigger tolerance against biotic/abiotic stresses and growth enhancement in plants. In this study, an endophytic bacterial strain from rapeseed was isolated to assess its role in enhancing plant growth and tolerance to abiotic stresses, as well as banded leaf and sheath blight disease in maize. Based on 16S rDNA and BIOLOG test analysis, the 330-2 strain was identified as Bacillus subtilis. The strain produced indole-3-acetic acid, siderophores, lytic enzymes and solubilized different sources of organic/inorganic phosphates and zinc. Furthermore, the strain strongly suppressed the in vitro growth of Rhizoctonia solani AG1-IA, Botrytis cinerea, Fusarium oxysporum, Alternaria alternata, Cochliobolus heterostrophus, and Nigrospora oryzae. The strain also significantly increased the seedling growth (ranging 14–37%) of rice and maize. Removing PCR analysis indicated that 114 genes were differentially expressed, among which 10%, 32% and 10% were involved in antibiotic production (e.g., srfAA, bae, fen, mln, and dfnI), metabolism (e.g., gltA, pabA, and ggt) and transportation of nutrients (e.g., fhu, glpT, and gltT), respectively. In summary, these results clearly indicate the effectiveness and mechanisms of B. subtilis strain 330-2 in enhancing plant growth, as well as tolerance to biotic/abiotic stresses, which suggests that the strain has great potential for commercialization as a vital biological control agent.

The biological clock affects aging through ras-1 (bd) and lag-1, and these two longevity genes together affect a clock phenotype and the clock oscillator in Neurospora crassa. Using an automated cell-counting technique for measuring conidial longevity, we show that the clock-associated genes lag-1 and ras-1 (bd) are true chronological longevity genes. For example, wild type (WT) has an estimated median life span of 24 days, while the double mutant lag-1, ras-1 (bd) has an estimated median life span of 120 days for macroconidia. We establish the biochemical function of lag-1 by complementing LAG1 and LAC1 in Saccharomyces cerevisiae with lag-1 in N. crassa. Longevity genes can affect the clock as well in that, the double mutant lag-1, ras-1 (bd) can stop the circadian rhythm in asexual reproduction (i.e., banding in race tubes) and lengthen the period of the frequency oscillator to 41 h. In contrast to the ras-1 (bd), lag-1 effects on chronological longevity, we find that this double mutant undergoes replicative senescence (i.e., the loss of replication function with time), unlike WT or the single mutants, lag-1 and ras-1 (bd). These results support the hypothesis that sphingolipid metabolism links aging and the biological clock through a common stress response.

In this study, we describe a novel mycovirus isolated from Ustilaginoidea virens, which was designated Ustilaginoidea virens nonsegmented virus 1 (UvNV-1). The sequence analysis revealed that UvNV-1 has two open reading frames (ORFs). ORF1 encodes an unknown protein, which is similar to the hypothetical protein BN7_5177 of Wickerhamomyces ciferrii. ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp), which is most closely related to Bryopsis mitochondria-associated dsRNA (BDRM) and is likely expressed by a +1 ribosomal frameshift within the sequence CCC_UUU_CGA. The phylogenetic analysis of the RdRp of UvNV-1 showed that UvNV-1 represents a new virus taxon of mycoviruses with a partitivirus-like lineage that is classified into the family of picorna-like viruses. Based on northern hybridization, UvNV-1 was found to be common to U. virens from different geographic locations in China. The biological comparison of virus-free and infected fungal strains revealed that UvNV-1 is likely to be cryptic to its host.

Yield losses caused by lodging in barley can be partially controlled by reducing plant height. In order to understand dwarfing mechanisms and efficiently use new dwarf germplasms for a breeding program, it is important to identify QTL of plant height components. QTL analysis was performed for seven plant height component traits using a DH population of 122 lines derived from the cross of Huaai 11 x Huadamai 6. Composite interval mapping procedures detected 20 QTL, which were mapped onto chromosomes 2H, 3H, 5H, 6H, and 7H. Eleven QTL were detected in 3 years and four QTL were detected in 2 years. QTL controlling all seven plant height component traits were found near the dwarfing gene btwd1 on chromosome 7H. These QTL accounted for 27.19 to 59.73% of phenotypic variation in seven plant height component traits. Positive transgressive segregation was found for all traits. Some of the QTL identified in this study will be useful for understanding the dwarfing mechanism and for developing new dwarf varieties using marker-assisted selection.

Rhizoctonia solani is a causative agent of sheath blight, which results in huge economic losses every year. During its life cycle, the formation of sclerotia helps Rhizoctonia solani withstand a variety of unfavorable factors. Oxidative stress is a key factor that induces sclerotium formation. The differentiated and undifferentiated phenotypes of R. solani AG-1-IA were obtained by controlling aerial conditions. Metabolomics based on the mass spectrometry technique combined with multivariate and univariate analyses was used to investigate the metabolic variation in vegetative, differentiated and undifferentiated mycelia. Our results revealed that during maturation, the metabolic levels of N2-acetyl-L-ornithine, 3,1'-(OH)2-Gamma-carotene, (5Z,7E)-(1S,3R)-24,24-difluoro-24a-homo-9,10-seco-5,7,10(19)-cholestatrien-1,3,25-triol, stoloniferone O, PA(O-18:0/12:0), PA(P-16:0/14:0), PA(P-16:0/16:(19Z)) and PA(P-16:0/17:2(9Z,12Z)) were suppressed in both differentiated and undifferentiated mycelia. The concentrations of PE(20:1(11Z)/14:1(9Z)), PE(P-16:0/20:4(5Z,8Z,11Z,13E)(15OH[S])) and PS(12:0/18:1(9Z)) were increased in the differentiated group, while increased levels of N(gamma)-nitro-L-arginine, tenuazonic acid and 9S,10S,11R-trihydroxy-12Z,15Z-octadecadienoic acid were found in the undifferentiated group. Our results suggest that different levels of these metabolites may act as biomarkers for the developmental stages of R. solani AG-1-IA. Moreover, the mechanisms of sclerotium formation and mycelium differentiation were elucidated at the metabolic level.

From the plant pathogen Ustilaginoidea virens, four double-stranded RNA (dsRNA) segments designated Uv-dsRNA1, -2, -3, and -4 were isolated, cloned, and sequenced. Uv-dsRNA1 (1775 bp) and -2 (1588 bp) potentially encode an RNA-dependent RNA polymerase (RdRp) and a viral coat protein (CP), respectively. Since the RdRp and CP sequences encoded by Uv-dsRNA1 and -2, respectively, are most closely related to, but clearly distinct from, those of viruses of the genus Partitivirus, they appear to be the two genome segments of a new partitivirus, for which the name Ustilaginoidea virens partitivirus 1 is proposed. In contrast, Uv-dsRNA3 (1352 bp) did not share significant sequence similarity with GenBank sequences, and the ORF of Uv-dsRNA4 (1119 bp) was only 32 % identical to a functionally unknown protein (GaRVMS2s3gp1) encoded by Gremmeniella abietina RNA virus MS2.

Ustilaginoidea virens is the causal agent of a serious disease of rice. Here, we report the presence of five dsRNA bands ranging from about 1.2, 1.5, 1.7, and 1.8 to 5.6 kb in an isolate of this fungus from China and the complete sequence of the largest dsRNA segment, putatively representing the genome of a novel virus, designated as Ustilaginoidea virens RNA virus 1 (UvRV1), UvRV1, which has a genome length of 5567 bp and has two consecutive open reading frames (ORFs) with a five-nucleotide overlap. Phylogenetic analysis showed that UvRV1 belongs to the genus of Victorivirus in the family Totiviridae. Electronic supplementary material The online version of this article (doi:10.1007/s00705-013-1615-9) contains supplementary material, which is available to authorized users.

There are increasing reports of geminivirus mixed infections of field plant hosts. These mixed infections have been suggested to result in recombinations, emergence of new viruses and new disease epidemics. We previously reported the occurrence of mixed infection between African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) resulting in severe symptoms in cassava fields in Cameroon. Here, we show that reassortment of DNA-A and DNA-B components of ACMV and EACMCV does not form viable recombinants. However, in the presence of both components of either virus, the DNA-A component of the other virus replicated and spread in the absence of its DNA-B component. This result suggests that failure of ACMV and EACMCV to form viable recombinants is due to the inability of each DNA-A component to trans-replicate the heterologous DNA-B component. This study also shows that ACMV DNA-A induces a resistance to ACMV and EACMCV as indicated by absence or late symptom development. Moreover, this resistance enabled plants to recover from severe symptoms caused by EACMCV in Nicotiana benthamiana, suggesting that the resistance induced is not specific to ACMV and is consistent with the phenomenon of cross-protection between related viruses.

To explore the pathogenesis of Rhizoctonia solani and its phytotoxin phenylacetic acid (PAA) on maize leaves and sheaths, treated leaf and sheath tissues were analyzed and interpreted by ultra-performance liquid chromatography-mass spectrometry combined with chemometrics. The PAA treatment had similar effects to those of R. solani on maize leaves regarding the metabolism of traumatin, phytosphingosine, vitexin 2'' O-beta-D-glucoside, rutin and DIBOA-glucoside, which were up-regulated, while the synthesis of OPC-8:0 and 12-OPDA, precursors for the synthesis of jasmonic acid, a plant defense signaling molecule, was down-regulated under both treatments. However, there were also discrepancies in the influences exhibited by R. solani and PAA as the metabolic concentration of zeaxanthin diglucoside in the R. solani infected leaf group decreased. Conversely, in the PAA-treated leaf group, the synthesis of zeaxanthin diglucoside was enhanced. Moreover, although the synthesis of 12 metabolites were suppressed in both the R. solani- and PAA-treated leaf tissues, the inhibitory effect of R. solani was stronger than that of PAA. An increased expression of quercitrin and quercetin 3-O-glucoside was observed in maize sheaths treated by R. solani, while their concentrations were not changed significantly in the PAA-treated sheaths. Furthermore, a significant decrease in the concentration of L-Glutamate, which plays important roles in plant resistance to necrotrophic pathogens, only occurred in the R. solani-treated sheath tissues. The differentiated metabolite levels may be the partial reason of why maize sheaths were more susceptible to R. solani than leaves and may explain the underlying mechanisms of R. solani pathogenesis.

Nonhost resistance protects plants against attack by the vast majority of potential pathogens, including phytopathogenic fungi. Despite its high biological importance, the molecular architecture of nonhost resistance has remained largely unexplored. Here, we describe the transcriptional responses of one particular genotype of barley (Hordeum vulgare subsp. vulgare ‘Ingrid’) to three different pairs of adapted (host) and nonadapted (nonhost) isolates of fungal pathogens, which belong to the genera Blumeria (powdery mildew), Puccinia (rust), and Magnaporthe (blast). Nonhost resistance against each of these pathogens was associated with changes in transcript abundance of distinct sets of nonhost-specific genes, although general (not nonhost-associated) transcriptional responses to the different pathogens overlapped considerably. The powdery mildew- and blast-induced differences in transcript abundance between host and nonhost interactions were significantly correlated with differences between a near-isogenic pair of barley lines that carry either the Mlo wild-type allele or the mutated mlo5 allele, which mediates basal resistance to powdery mildew. Moreover, during the interactions of barley with the different host or nonhost pathogens, similar patterns of overrepresented and underrepresented functional categories of genes were found. The results suggest that nonhost resistance and basal host defense of barley are functionally related and that nonhost resistance to different fungal pathogens is associated with more robust regulation of complex but largely nonoverlapping sets of pathogen-responsive genes involved in similar metabolic or signaling pathways.

A major challenge of systems biology is explaining complex traits, such as the biological clock, in terms of the kinetics of macromolecules. The clock poses at least four challenges for systems biology: (i) identifying the genetic network to explain the clock mechanism quantitatively; (ii) specifying the clock's functional connection to a thousand or more genes and their products in the genome; (iii) explaining the clock's response to light and other environmental cues; and (iv) explaining how the clock's genetic network evolves. Here, the authors illustrate an approach to these problems by fitting an ensemble of genetic networks to microarray data derived from oligonucleotide arrays with approximately all 11 000 Neurospora crassa genes represented. A promising genetic network for the clock mechanism is identified.

The products of five structural genes and two regulatory genes of the qa gene cluster of Neurospora crassa control the metabolism of quinic acid (QA) as a carbon source. A detailed genetic network model of this metabolic process has been reported. This investigation is designed to expand the current model of the QA reaction network. The ensemble method of network identification was used to model RNA profiling data on the qa gene cluster. Through microarray and cluster analysis, genome-wide identification of RNA transcripts associated with quinic acid metabolism in N. crassa is described and suggests a connection to other metabolic circuits. More than 100 genes whose products include carbon metabolism, protein degradation and modification, amino acid metabolism and ribosome synthesis appear to be connected to quinic acid metabolism. The core of the qa gene cluster network is validated with respect to RNA profiling data obtained from microarrays.

To study protein ubiquitination pathways in the interaction of barley (Hordeum vulgare) with the powdery mildew fungus (Blumeria graminis), we measured protein turnover and performed transient-induced gene silencing (TIGS) of ubiquitin and 26S proteasome subunit encoding genes in epidermal cells. Attack by B. graminis hyperdestabilized a novel unstable green fluorescent protein fusion that contains a destabilization domain of a putative barley 1-aminocyclopropane-1-carboxylate synthase, suggesting enhanced protein turnover. Partial depletion of cellular ubiquitin levels by TIGS induced extreme susceptibility of transformed cells toward the appropriate host pathogen B. graminis f. sp hordei, whereas papilla-based resistance to the nonhost pathogen B. graminis f. sp tritici and host resistance mediated by the mlo gene (for mildew resistance locus O) remained unaffected. Cells were rescued from TIGS-induced ubiquitin depletion by synthetic genes encoding wild-type or mutant barley monoubiquitin proteins. The strongest rescue was from a gene encoding a K63R mutant form of ubiquitin blocked in several ubiquitination pathways while still allowing Lys-48–dependent polyubiquitination required for proteasomal protein degradation. Systematic RNA interference of 40 genes encoding all 17 subunits of the proteasome 19S regulatory particle failed to induce hypersusceptibility against B. graminis f. sp hordei. This suggests a role for Lys-48–linked protein polyubiquitination, which is independent from the proteasome pathway, in basal host defense of barley.

A novel removing polymerase chain reaction (R-PCR) technique was developed, which can eliminate undesired genes, cycle by cycle, with efficiencies of 60.9% (cDNAs), 73.6% (genomic DNAs), and ~ 100% (four DNA fragments were tested). Major components of the R-PCR include drivers, a thermostable restriction enzyme - ApeKI, and a poly(dA) adapter with mismatched restriction enzyme recognition sites. Drivers were generated from the undesired genes. In each cycle of R-PCR, drivers anneal to complementary sequences and allow extension by Taq DNA polymerase. Thus, ApeKI restriction sites in the undesired genes are recovered, and adapters of these undesired DNA fragments are removed. Using R-PCR, we isolated maize upregulated defense-responsive genes and Blumeria graminis specialized genes, including key pathogenesis-related effectors. Our results show that after the R-PCR reaction, most undesired genes, including very abundant genes, became undetectable. The R-PCR is an easy and cost-efficient method to eliminate undesired genes and clone desired genes.

An ensemble of genetic networks that describe how the model fungal system, Neurospora crassa, utilizes quinic acid (QA) as a sole carbon source has been identified previously. A genetic network for QA metabolism involves the genes, qa-1F and qa-1S, that encode a transcriptional activator and repressor, respectively and structural genes, qa-2, qa-3, qa-4, qa-x, and qa-y. By a series of 4 separate and independent, model-guided, microarray experiments a total of 50 genes are identified as QA-responsive and hypothesized to be under QA-1F control and/or the control of a second QA-responsive transcription factor (NCU03643) both in the fungal binuclear Zn(II)2Cys6 cluster family. QA-1F regulation is not sufficient to explain the quantitative variation in expression profiles of the 50 QA-responsive genes. QA-responsive genes include genes with products in 8 mutually connected metabolic pathways with 7 of them one step removed from the tricarboxylic (TCA) Cycle and with 7 of them one step removed from glycolysis: (1) starch and sucrose metabolism; (2) glycolysis/glucanogenesis; (3) TCA Cycle; (4) butanoate metabolism; (5) pyruvate metabolism; (6) aromatic amino acid and QA metabolism; (7) valine, leucine, and isoleucine degradation; and (8) transport of sugars and amino acids. Gene products both in aromatic amino acid and QA metabolism and transport show an immediate response to shift to QA, while genes with products in the remaining 7 metabolic modules generally show a delayed response to shift to QA. The additional QA-responsive cutinase transcription factor-1β (NCU03643) is found to have a delayed response to shift to QA. The series of microarray experiments are used to expand the previously identified genetic network describing the qa gene cluster to include all 50 QA-responsive genes including the second transcription factor (NCU03643). These studies illustrate new methodologies from systems biology to guide model-driven discoveries about a core metabolic network involving carbon and amino acid metabolism in N. crassa.