The E3 Ubiquitin Ligase ATL9 Affects Expression of Defense Related Genes, Cell Death and Callose Deposition in Response to Fungal Infection

Plants use diverse strategies to defend themselves from biotic stresses in nature, which include the activation of defense gene expression and a variety of signal transduction pathways. Previous studies have shown that protein ubiquitination plays a critical role in plant defense responses, however the details of its function remain unclear. Our previous work has shown that increasing expression levels of ATL9, an E3 ubiquitin ligase in Arabidopsis thaliana, increased resistance to infection by the fungal pathogen, Golovinomyces cichoracearum. In this study, we demonstrate that the defense-related proteins PDF1.2, PCC1 and FBS1 directly interact with ATL9 and are targeted for degradation to the proteasome by ATL9. The expression levels of PDF1.2, PCC1 and FBS1 are decreased in T-DNA insertional mutants of atl9 and T-DNA insertional mutants of pdf1.2, pcc1 and fbs1 are more susceptible to fungal infection. In addition, callose is more heavily deposited at infection sites in the mutants of atl9, fbs1, pcc1 and pdf1.2. Overexpression of ATL9 and of mutants in fbs1, pcc1 and pdf1.2 showed increased levels of cell death during infection. Together these results indicate that ubiquitination, cell death and callose deposition may work together to enhance defense responses to fungal pathogens.

The Mla Pathway Plays an Essential Role in the Intrinsic Resistance ofBurkholderia cepaciaComplex Species to Antimicrobials and Host Innate Components

ABSTRACTAntibiotic resistance is a threat to our modern society, and new strategies leading to the identification of new molecules or targets to combat multidrug-resistant pathogens are needed. Species of the genusBurkholderia, including theBurkholderia cepaciacomplex (Bcc),Burkholderia pseudomallei, andBurkholderia mallei, can be highly pathogenic and are intrinsically resistant to multiple classes of antibiotics. Bcc species are nonetheless sensitive to extracellular products released byPseudomonas aeruginosain interspecies competition. We screened forBurkholderiatransposon mutants with increased sensitivity toP. aeruginosaspent medium and identified multiple mutants in genes sharing homology with the Mla pathway. Insertional mutants in representative genes of the Bcc Mla pathway had a compromised cell membrane and were more sensitive to various extracellular stresses, including antibiotics and human serum. More precisely,mlamutants in the Bcc speciesBurkholderia cenocepaciaandBurkholderia dolosawere more susceptible to Gram-positive antibiotics (i.e., macrolides and rifampin), fluoroquinolones, tetracyclines, and chloramphenicol. Genetic complementation ofmlaCinsertional mutants restored cell permeability and resistance to Gram-positive antibiotics. Importantly, Bccmlamutants were not universally weaker strains since their susceptibilities to other classes of antibiotics were unaffected. Although cell permeability of homologousmlamutants inEscherichia coliorP. aeruginosawas also impaired, they were not more sensitive to Gram-positive antibiotics or other antimicrobials as was observed in Bccmlamutants. Together, the data suggest that the Mla pathway inBurkholderiamay play a different biological role, which could potentially represent aBurkholderia-specific drug target in combination therapy with antibiotic adjuvants.IMPORTANCEThe outer membrane of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore compromising this structure could increase sensitivity to currently available antibiotics. In this study, we show that the Mla pathway, a system involved in maintaining the integrity of the outer membrane, is genetically and functionally different inBurkholderia cepaciacomplex species compared to that in other proteobacteria. Mutants inmlagenes ofBurkholderia cenocepaciaorBurkholderia dolosawere sensitive to Gram-positive antibiotics, while this effect was not observed inEscherichia coliorPseudomonas aeruginosa. The Mla pathway inBurkholderiaspecies may represent an ideal genus-specific target to address their intrinsic antimicrobial resistances.

Bidirectional-Genetics Platform, a Dual-Purpose Mutagenesis Strategy for Filamentous Fungi

ABSTRACTRapidly increasing fungal genome sequences call for efficient ways of generating mutants to translate quickly gene sequences into their functions. A reverse genetic strategy via targeted gene replacement (TGR) has been inefficient for many filamentous fungi due to dominant production of undesirable ectopic transformants. Although large-scale random insertional mutagenesis via transformation (i.e., forward genetics) facilitates high-throughput uncovering of novel genes of interest, generating a huge number of transformants, which is necessary to ensure the likelihood of mutagenizing most genes, is time-consuming. We propose a new strategy, entitled theBidirectional-Genetics (BiG) platform, which combines both forward and reverse genetic strategies by recycling ectopic transformants derived from TGR as a source for random insertional mutants. The BiG platform was evaluated using the rice blast fungusMagnaporthe oryzaeas a model. Over 10% of >1,000M. oryzaeectopic transformants, generated during disruption of specific genes, displayed abnormality in vegetative growth, pigmentation, and/or asexual reproduction. In this pool of putative mutants, we isolated insertional mutants with mutations in three genes involved in histidine biosynthesis (MoHIS5), vegetative growth (MoVPS74), or conidiophore formation (MoFRQ) (where “Mo” indicates “M. oryzae”), supporting the utility of this platform for systematic gene function studies.