Characterization of Z/E11- and Z9-desaturases from the obliquebanded leafroller moth, Choristoneura rosaceana

A ▵11-desaturase gene was cloned from the sex pheromone gland of the obliquebanded leafroller moth, Choristoneura rosaceana. The desaturase cDNA sequence spans 1300 nucleotides with an open reading frame encoding a 335 amino-acid protein, which has 81% identity to a Z/E11-desaturase of the redbanded leafroller moth, Argyrotaenia velutinana. A functional assay with a pYES2 yeast expression system demonstrated that the ▵11-desaturase exhibits unusual substrate and stereospecificities in producing a Z/E11 mixture (7:1) of only C14 acids. A metabolic Z9-desaturase also was cloned from fat body of this species, and proved to be in the class that produces more Z9-16:Acid than Z9-18:Acid

A gene cluster in Agrobacterium vitis homologous to polyketide synthase operons is associated with grape necrosis and hypersensitive response induction on tobacco

Here, we identify a cluster of eight genes on chromosome 2 of Agrobacterium vitis that is associated with the ability of the bacterium to cause a hypersensitive response on tobacco and a necrosis of grape shoot explants. Three of these genes share a high level of structural and sequence similarity to clusters of genes in other bacteria that encode the enzymes for biosynthesis of polyketides and long-chain polyunsaturated fatty acids. No similar gene clusters were discovered in sequenced genomes of other members of Rhizobiale

Characterization of a Fusarium graminearum Salicylate Hydroxylase

Salicylic acid (SA) plays an important role in regulating plant defense responses against pathogens. However, pathogens have evolved ways to manipulate plant SA-mediated defense signaling. Fusarium graminearum causes Fusarium head blight (FHB) and reduces crop yields and quality by producing various mycotoxins. In this study, we aimed to identify the salicylate hydroxylase in F. graminearum and determine its role in wheat head blight development. We initially identified a gene in F. graminearum strain NRRL 46422 that encodes a putative salicylate hydroxylase (designated FgShyC). However, the FgShyC deletion mutant showed a similar ability to degrade SA as wild-type strain 46422; nor did overexpression of FgShyC in E. coli convert SA to catechol. The results indicate that FgShyC is not involved in SA degradation. Further genome sequence analyses resulted in the identification of eight salicylate hydroxylase candidates. Upon addition of 1 mM SA, FGSG_03657 (designated FgShy1), was induced approximately 400-fold. Heterologous expression of FgShy1 in E. coli converted SA to catechol, confirming that FgShy1 is a salicylate hydroxylase. Deletion mutants of FgShy1 were greatly impaired but not completely blocked in SA degradation. Expression analyses of infected tissue showed that FgShy1 was induced during infection, but virulence assays revealed that deletion of FgShy1 alone was not sufficient to affect FHB severity. Although the Fgshy1 deletion mutant did not reduce pathogenicity, we cannot rule out that additional salicylate hydroxylases are present in F. graminearum and characterization of these enzymes will be necessary to fully understand the role of SA-degradation in FHB pathogenesis

Detoxification and Excretion of Trichothecenes in Transgenic Arabidopsis thaliana Expressing Fusarium graminearum Trichothecene 3-O-acetyltransferase

Fusarium graminearum, the causal agent of Fusarium head blight (FHB), produces trichothecenes including deoxynivalenol (DON), nivalenol (NIV), and 3,7,15-trihydroxy-12,13-epoxytrichothec-9-ene (NX-3). These toxins contaminate grains and cause profound health problems in humans and animals. To explore exploiting a fungal self-protection mechanism in plants, we examined the ability of F. graminearum trichothecene 3-O-acetyltransferase (FgTri101) to detoxify several key trichothecenes produced by F. graminearum: DON, 15-ADON, NX-3, and NIV. FgTri101 was cloned from F. graminearum and expressed in Arabidopsis plants. We compared the phytotoxic effects of purified DON, NIV, and NX-3 on the root growth of transgenic Arabidopsis expressing FgTri101. Compared to wild type and GUS controls, FgTri101 transgenic Arabidopsis plants displayed significantly longer root length on media containing DON and NX-3. Furthermore, we confirmed that the FgTri101 transgenic plants acetylated DON to 3-ADON, 15-ADON to 3,15-diADON, and NX-3 to NX-2, but did not acetylate NIV. Approximately 90% of the converted toxins were excreted into the media. Our study indicates that transgenic Arabidopsis expressing FgTri101 can provide plant protection by detoxifying trichothecenes and excreting the acetylated toxins out of plant cells. Characterization of plant transporters involved in trichothecene efflux will provide novel targets to reduce FHB and mycotoxin contamination in economically important plant crops

Regulation of Long-Chain N-Acyl-Homoserine Lactones in Agrobacterium vitis

Homologs of quorum-sensing luxR and luxI regulatory genes, avsR and avsI, were identified in Agrobacterium vitis strain F2/5. Compared to other LuxI proteins from related species, the deduced AvsI shows the greatest identity to SinI (71%) from Sinorhizobium meliloti Rm1021. AvsR possesses characteristic autoinducer binding and helix-turn-helix DNA binding domains and shares a high level of identity with SinR (38%) from Rm1021. Site-directed mutagenesis of avsR and avsI was performed, and both genes are essential for hypersensitive-like response (HR) and necrosis. Two hypothetical proteins (ORF1 and ORF2) that are positioned downstream of avsR-avsI are also essential for the phenotypes. Profiles of N-acyl-homoserine lactones (AHLs) isolated from the wild type and mutants revealed that disruption of avsI, ORF1, or ORF2 abolished the production of long-chain AHLs. Disruption of avsR reduces long-chain AHLs. Expression of a cloned avsI gene in A. tumefaciens strain NT1 resulted in synthesis of long-chain AHLs. The necrosis and HR phenotypes of the avsI and avsR mutants were fully complemented with cloned avsI. The addition of synthetic AHLs (C(16:1) and 3-O-C(16:1)) complemented grape necrosis in the avsR, avsI, ORF1, and ORF2 mutants. It was determined by reverse transcriptase PCR that the expression level of avsI is regulated by avsR but not by aviR or avhR, two other luxR homologs which were previously shown to be associated with induction of a tobacco hypersensitive response and grape necrosis. We further verified that avsR regulates avsI by measuring the expression of an avsI::lacZ fusion construct

Transgenic expression of antimicrobial peptide D2A21 confers resistance to diseases incited by Pseudomonas syringae pv. tabaci and Xanthomonas citri, but not Candidatus Liberibacter asiaticus.

Citrus Huanglongbing (HLB) associated with 'Candidatus Liberibacter asiaticus' (Las) and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs) in vitro for their potential application in genetic engineering. D2A21 was one of the most active AMPs against X. citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation and D2A21 expression was confirmed by molecular analysis. We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by PCR and RT-qPCR. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with X. citri 3213. Transgenic Carrizo plants were challenged for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants. However, our results showed that transgenic plants expressing D2A21 did not significantly reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri but not Las. Our results underscore the difficulty in controlling HLB compared to other bacterial diseases

‘Candidatus Liberibacter asiaticus’ Encodes Two Novel Autotransporters that Target to Mitochondria

As a phloem-limited, intracellular bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (Las) has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide.  In this study, we characterized two novel autotransporter proteins of Las, and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of the signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria. These findings may lead to a better understanding of the pathogenesis of this intracellular “energy parasitic” bacterium, and to more efficient characterizing new molecular targets for HLB control

luxR Homolog avhR in Agrobacterium vitis Affects the Development of a Grape-Specific Necrosis and a Tobacco Hypersensitive Response

The luxR homolog aviR in Agrobacterium vitis strain F2/5 was recently shown to be associated with induction of a hypersensitive response (HR) on tobacco and necrosis on grape plants, indicating that the responses are regulated by quorum sensing. We now report a second luxR homolog, avhR, whose disruption (mutant M1320) results in HR-negative and reduced grape necrosis phenotypes. The deduced AvhR protein has characteristic autoinducer binding and DNA binding domains and is unique among reported functional LuxR homologs in having substitutions at highly conserved Asp70, Trp57, and Trp85 residues, which are predicted to play important roles in autoinducer binding in TraR. M1320 was fully complemented with cloned avhR. The same array of N-acylhomoserine lactones (AHL) from F2/5, M1320, and complemented M1320 were observed; however, the signal strength from extracts of 6-day-old M1320 cultures was stronger than that of F2/5. Cultures of F2/5 amended with AHL extracts from overnight and 6-day cultures of F2/5 and M1320 were not affected in ability to cause HR or necrosis. A region of about 14 kb flanking avhR was sequenced and compared with homologous regions of A. tumefaciens C58 and Sinorhizobium meliloti Rm1021 genomes. Gene order and homology are conserved between the species. A site-directed mutation in a putative gene that resides downstream of avhR and that has homology to genes belonging to the ATP-binding cassette transporter family did not affect HR or necrosis phenotypes. It was determined that avhR and aviR are expressed independently and that neither regulates the expression of a clpA homolog in F2/5