Specific and Accurate Detection of the Citrus Greening Pathogen <em>Candidatus liberibacter </em> spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

University of South Carolina

AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.

RNA-Seq Links the Transcription Factors AINTEGUMENTA and AINTEGUMENTA-LIKE6 to Cell Wall Remodeling and Plant Defense Pathways.

The transcriptional regulator NPR1 mediates salicylic acid (SA)-induced plant immunity. NPR1 is also required for tolerance to high concentrations of SA. NPR1-interacting protein, NIMIN1, represses immune response by interacting with and negating NPR1. We tested the salicylic acid tolerance of transgenic plants overexpressing NIMIN1 and found that these plants displayed SA intolerance, similar to the npr1 mutant, due to sequestration of NPR1 by NIMIN1. Plants overexpressing mutated NIMIN1 that cannot interact with NPR1 showed no SA tolerance defect. Gene expression analysis showed that NPR1 is required for SA-stress induced as well as pathogen-induced NIMIN1 expression. These results indicate that over-accumulation of a negative regulator renders plants hypersensitive to SA by limiting NPR1 function. Furthermore, NPR1 activates negative regulators such as NIMIN1 for feedback inhibition of SA signaling to maintain immune homeostasis.

Overexpression of Arabidopsis NIMIN1 results in salicylate intolerance.

Sugar, as a signal molecule, has significant functions in signal transduction in which the seven-transmembrane regulator of G-protein signaling (RGS1) protein participates. D-Glucose causes endocytosis of the AtRGS1, leading to the physical uncoupling of AtRGS1 from AtGPA1 and thus a release of the GAP activity and concomitant sustained activation of G-protein signaling. Autophagy involves in massive degradation and recycling of cytoplasmic components to survive environmental stresses. The function of autophagy in AtRGS1 endocytosis during D-glucose stimulation has not been elucidated. In this study, we investigate the relationship between autophagy and AtRGS1 in response to D-glucose. Our findings demonstrated that AtRGS1 mediated the activation of autophagy by affecting the activities of the five functional groups of protein complexes and promoted the formation of autophagosomes under D-glucose application. When the autophagy pathway was interrupted, AtRGS1 recovery increased and endocytosis of ATRGS1 was inhibited, indicating that autophagy pathway plays an important role in regulating the endocytosis and recovery of AtRGS1 after D-glucose stimulation.

Endocytosis of AtRGS1 Is Regulated by the Autophagy Pathway after D-Glucose Stimulation.

 is a Gram-negative, environmentally ubiquitous bacterium that produces a secondary metabolite, called heat-stable antifungal factor (HSAF), as an antifungal factor against plant and animal fungal pathogens. 4-Hydroxybenzoic acid (4-HBA) is a newly identified diffusible factor that regulates HSAF synthesis via  LysR (LysR), an LysR-type transcription factor (TF). Here, to identify additional TFs within the 4-HBA regulatory pathway that control HSAF production, we reanalyzed the LenB2-based transcriptomic data, in which LenB2 is the enzyme responsible for 4-HBA production. This survey led to identification of three TFs (Le4806, Le4969, and Le3904). Of them, LarR (Le4806), a member of the MarR family proteins, was identified as a new TF that participated in the 4-HBA-dependent regulation of HSAF production. Our data show the following: (i) that LarR is a downstream component of the 4-HBA regulatory pathway controlling the HSAF level, while LysR is the receptor of 4-HBA; (ii) that 4-HBA and LysR have opposite regulatory effects on  transcription whereby  transcript is negatively modulated by 4-HBA while LysR, in contrast, exerts positive transcriptional regulation by directly binding to the  promoter without being affected by 4-HBA ; (iii) that LarR, similar to LysR, can bind to the promoter of the HSAF biosynthetic gene operon, leading to positive regulation of HSAF production; and (iv) that LarR and LysR cannot interact and instead control HSAF biosynthesis independently. These results outline a previously uncharacterized mechanism by which biosynthesis of the antibiotic HSAF in  is modulated by the interplay of 4-HBA, a diffusible molecule, and two different TFs. Bacteria use diverse chemical signaling molecules to regulate a wide range of physiological and cellular processes. 4-HBA is an "old" chemical molecule that is produced by diverse bacterial species, but its regulatory function and working mechanism remain largely unknown. We previously found that 4-HBA in  could serve as a diffusible factor regulating HSAF synthesis via LysR Here, we further identified LarR, an MarR family protein, as a second TF that participates in the 4-HBA-dependent regulation of HSAF biosynthesis. Our results dissected how LarR acts as a protein linker to connect 4-HBA and HSAF synthesis, whereby LarR also has cross talk with LysR Thus, our findings not only provide fundamental insight regarding how a diffusible molecule (4-HBA) adopts two different types of TFs for coordinating HSAF biosynthesis but also show the use of applied microbiology to increase the yield of the antibiotic HSAF by modification of the 4-HBA regulatory pathway in .

Heat-Stable Antifungal Factor (HSAF) Biosynthesis in Lysobacter enzymogenes Is Controlled by the Interplay of Two Transcription Factors and a Diffusible Molecule.

Heat-stable antifungal factor (HSAF) is a polycyclic tetramate macrolactam secondary metabolite that exhibits broad-spectrum inhibitory activities against filamentous fungal pathogens. The native yield of this chemical is low. It is also a great challenge to synthesize HSAF artificially, due to its complex structure. Understanding the regulatory mechanism underlying HSAF biosynthesis could provide genetic basis for engineering high HSAF-producing strain. The transcription factor Clp is a global regulator that controls bacterial pathogenicity and the expression of one hundred related genes in the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc). Diffusible signal factor (DSF) chemical signaling is the only well-characterized upstream regulatory pathway that involves downstream Clp regulation in Xcc. Such a regulatory hierarchy between DSF signaling and Clp is also conserved in the Gram-negative biological control agent Lysobacter enzymogenes, where the DSF signaling system controls antifungal antibiotic HSAF biosynthesis via Clp.

LesR is a novel upstream regulator that controls downstream Clp expression to modulate antibiotic HSAF biosynthesis and cell aggregation in Lysobacter enzymogenes OH11.

Most plant bacterial pathogens rely on type III effectors to cause diseases. Although it is well known that the plant hormone salicylic acid (SA) plays an essential role in defense, whether the master regulator of SA signaling, NPR1, is targeted by any plant pathogen effectors is unknown. SA facilitates the reduction of cytosolic NPR1 oligomers into monomers, which enter the nucleus and function as transcriptional coactivators of plant defense genes. We show that SA promotes the interaction between the Pseudomonas syringae type III effector AvrPtoB and NPR1. In the presence of SA, AvrPtoB mediates the degradation of NPR1 via the host 26S proteasome in a manner dependent on AvrPtoB's E3 ligase activity. Intriguingly, we found that NPR1 plays an important role in MAMP-triggered immunity (MTI), inducing the expression of MTI marker genes. Thus, this work uncovers a strategy in which AvrPtoB targets NPR1 and represses NPR1-dependent SA signaling, thereby subverting plant innate immunity.

A Bacterial Type III Effector Targets the Master Regulator of Salicylic Acid Signaling, NPR1, to Subvert Plant Immunity.

Xanthomonas oryzae pv. oryzicola is an X. oryzae pathovar that causes bacterial leaf streak in rice. In this study, we performed functional characterization of a nine-gene his operon in X. oryzae pv. oryzicola. Sequence analysis indicates that this operon is highly conserved in Xanthomonas spp. Auxotrophic assays confirmed that the his operon was involved in histidine biosynthesis. We found that two genes within this operon, trpR and hisB, were required for virulence and bacterial growth in planta. Further research revealed that trpR and hisB play different roles in X. oryzae pv. oryzicola. The trpR acts as a transcriptional repressor and could negatively regulate the expression of hisG, -D, -C, -B, -H, -A, and -F. hisB, which encodes a bifunctional enzyme implicated in histidine biosynthesis, was shown to be required for xanthomonadin production in X. oryzae pv. oryzicola. The disruption of hisB reduced the transcriptional expression of five known shikimate pathway-related genes xanB2, aroE, aroA, aroC, and aroK. We found that the his operon in X. oryzae pv. oryzicola is not involved in hypersensitive response in nonhost tobacco plants. Collectively, our results revealed that two genes in histidine biosynthesis operon play an important role in the pathogenicity of X. oryzae pv. oryzicola Rs105.

Zheng Qing Fu

Department of Biological Sciences

University of South Carolina

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Zheng Qing Fu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

University of South Carolina

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Department of Biological Sciences