Disruption of poly(ADP-ribosyl)ation mechanisms alters responses of Arabidopsis to biotic stress.

Poly(ADP-ribosyl)ation is a posttranslational protein modification in which ADP-ribose (ADP-Rib) units derived from NAD(+) are attached to proteins by poly(ADP-Rib) polymerase (PARP) enzymes. ADP-Rib groups are removed from these polymer chains by the enzyme poly(ADP-Rib) glycohydrolase (PARG). In animals, poly(ADP-ribosyl)ation is associated with DNA damage responses and programmed cell death. Previously, we hypothesized a role for poly(ADP-ribosyl)ation in plant defense responses when we detected defense-associated expression of the poly(ADP-ribosyl)ation-related genes PARG2 and NUDT7 and observed altered callose deposition in the presence of a chemical PARP inhibitor. The role of poly(ADP-ribosyl)ation in plant defenses was more extensively investigated in this study, using Arabidopsis (Arabidopsis thaliana). Pharmacological inhibition of PARP using 3-aminobenzamide perturbs certain innate immune responses to microbe-associated molecular patterns (flg22 and elf18), including callose deposition, lignin deposition, pigment accumulation, and phenylalanine ammonia lyase activity, but does not disrupt other responses, such as the initial oxidative burst and expression of some early defense-associated genes. Mutant parg1 seedlings exhibit exaggerated seedling growth inhibition and pigment accumulation in response to elf18 and are hypersensitive to the DNA-damaging agent mitomycin C. Both parg1 and parg2 knockout plants show accelerated onset of disease symptoms when infected with Botrytis cinerea. Cellular levels of ADP-Rib polymer increase after infection with avirulent Pseudomonas syringae pv tomato DC3000 avrRpt2(+), and pathogen-dependent changes in the poly(ADP-ribosyl)ation of discrete proteins were also observed. We conclude that poly(ADP-ribosyl)ation is a functional component in plant responses to biotic stress.

Discovery of ADP-ribosylation and other plant defense pathway elements through expression profiling of four different Arabidopsis-Pseudomonas R-avr interactions.

A dissection of plant defense pathways was initiated through gene expression profiling of the responses of a single Arabidopsis thaliana genotype to isogenic Pseudomonas syringae strains expressing one of four different cloned avirulence (avr) genes. Differences in the expression profiles elicited by different resistance (R)-avr interactions were observed. A role for poly(ADP-ribosyl)ation in plant defense responses was suggested initially by the upregulated expression of genes encoding NUDT7 and poly(ADP-ribose) glycohydrolase in multiple R-avr interactions. Gene knockout plant lines were tested for 20 candidate genes identified by the expression profiling, and Arabidopsis NUDT7 mutants allowed less growth of virulent P. syringae (as previously reported) but also exhibited a reduced hypersensitive-response phenotype. Inhibitors of poly(ADP-ribose) polymerase (PARP) disrupted FLS2-mediated basal defense responses such as callose deposition. EIN2 (ethylene response) and IXR1 and IXR2 (cellulose synthase) mutants impacted the FLS2-mediated responses that occur during PARP inhibition, whereas no impacts were observed for NPR1, PAD4, or NDR1 mutants. In the expression profiling work, false-positive selection and grouping of genes was reduced by requiring simultaneous satisfaction of statistical significance criteria for each of three separate analysis methods, and by clustering genes based on statistical confidence values for each gene rather than on average fold-change of transcript abundance.

Evidence that CTR1-mediated ethylene signal transduction in tomato is encoded by a multigene family whose members display distinct regulatory features.

Ethylene governs a range of developmental and response processes in plants. In Arabidopsis thaliana, the Raf-like kinase CTR1 acts as a key negative regulator of ethylene responses. While only one gene with CTR1 function apparently exists in Arabidopsis, we have isolated a family of CTR1- like genes in tomato ( Lycopersicon esculentum ). Based on amino acid alignments and phylogenetic analysis, these tomato CTR1- like genes are more similar to Arabidopsis CTR1 than any other sequences in the Arabidopsis genome. Structural analysis reveals considerable conservation in the size and position of the exons between Arabidopsis and tomato CTR1 genomic sequences. Complementation of the Arabidopsis ctr1-8 mutant with each of the tomato CTR genes indicates that they are all capable of functioning as negative regulators of the ethylene pathway. We previously reported that LeCTR1 expression is up-regulated in response to ethylene. Here, quantitative real-time PCR was carried out to detail expression for LeCTR1 and the additional CTR1 -like genes of tomato. Our results indicate that the tomato CTR1 gene family is differentially regulated at the mRNA level by ethylene and during stages of development marked by increased ethylene biosynthesis, including fruit ripening. The possibility of a multi-gene family of CTR1 -like genes in other species besides tomato was examined through mining of EST and genomic sequence databases.

LeCTR1, a tomato CTR1-like gene, demonstrates ethylene signaling ability in Arabidopsis and novel expression patterns in tomato.

LeCTR1 was initially isolated by both differential display reverse transcriptase-polymerase chain reaction screening for tomato (Lycopersicon esculentum) fruit ethylene-inducible genes and through homology with the Arabidopsis CTR1 cDNA. LeCTR1 shares strong nucleotide sequence homology with Arabidopsis CTR1, a gene acting downstream of the ethylene receptor and showing similarity to the Raf family of serine/threonine protein kinases. The length of the LeCTR1 transcribed region from ATG to stop codon (12,000 bp) is more than twice that of Arabidopsis CTR1 (4,700 bp). Structural analysis reveals perfect conservation of both the number and position of introns and exons in LeCTR1 and Arabidopsis CTR1. The introns in LeCTR1 are much longer, however. To address whether this structural conservation is indicative of functional conservation of the corresponding proteins, we expressed LeCTR1 in the Arabidopsis ctr1-1 (constitutive triple response 1) mutant under the direction of the 35S promoter. Our data clearly show that ectopic expression of LeCTR1 in the Arabidopsis ctr1-1 mutant can restore normal ethylene signaling. The recovery of normal ethylene sensitivity upon heterologous expression of LeCTR1 was also confirmed by restored glucose sensitivity absent in the Arabidopsis ctr1-1 mutant. Expression studies confirm ethylene responsiveness of LeCTR1 in various tissues, including ripening fruit, and may suggest the evolution of alternate regulatory mechanisms in tomato versus Arabidopsis.