HarpinPss, a proteinaceous elicitor from Pseudomonas syringae pv. syringae, is a glycine-rich, cysteine-lacking, heat-stable protein. It can elicit the hypersensitive response (HR) when delivered to the surface of plant cells. HRAP (hypersensitive response assisting protein) is an amphipathic protein purified from sweet pepper and could intensify harpinPss¡Vmediated HR in sweet pepper. In the previous research, harpinPss was present as monomer, dimer, trimer, tetramer, and ocatamer forms in neutral pH buffer. Only monomer and dimer forms of harpinPss induced hypersensitive response in nonhost plants. HRAP could cause multimeric forms of harpinPss dissociation into monomer forms. The interaction between HRAP and harpinPss is an important issue. HRAP contained three positively charged regions, a typical signal peptide and a cAMP-dependent phosphorylation site. In this study, these regions of HRAP would be truncated and identified whether these truncated HRAP fragments could promote harpinPss dissociation. Different combinations of truncated HRAP and harpinPss were used to identify the protein-interaction regions between two proteins. HarpinPss triggers HR via interaction with cAMP phosphorated region of HRAP and MAPK pathway transduction. When cAMP region of HRAP was truncated, harpinPss still triggers HR via polymerization and anchor on lipid bilayers to form an ion-conducting pore.

No description available.

In our investigation, fully expanded tobacco leaves were used to study the hypersensitive response caused by the inoculation of INF1 purified from E. coli strains DH5£\

Infection of fully expanded leaves of tobacco with INF1 causes the appearance of HR lesions within 12 h and progressive to all infection sites after 48 h treatment. Among the POD isozymes, the increase of cationic PODs and anionic PODs is correlated with the rise of lignin contents in INF1-treated leaves, especially cationic PODs (pI 9.5, pI 8.7, pI 8.3, pI 7.8, pI 7.4). It was suggested that the induction of POD activity resulted in part of H2O2 reduction. The increase of cationic (pI 9.5) and anionic (pI 4.4) POD transcripts was correlated with the increased cationic and anionic PODs activity in INF1-treated leaves. Therefore, the increased POD activity is due to the de novo synthesis of the cationic (pI 9.5) and anionic (pI 4.4) PODs in INF1-treated leaves. The increase in cationic pI 9.6 laccase transcript was also correlated with the increased cationic laccase activity in INF1-treated leaves. Our results suggest that laccase might play a major role on lignin biosynthesis at the early stage (6 h), and as the inoculation time was prolonged, peroxidases (especially cationic POD) and laccases will work together on lignin biosynthesis.

Harpinpss, a pathogenic protein, encoded by hrpZ in the hrp gene cluster from Pswudomonas syringae pv. syringae, can induce the hypersensitive response in tobacco (Nicotiana tabacum L. cv. Xanthi). The lesion area on the tobacco leaves was visible 6 h after inoculation with harpin, and was evident 12 h after inoculation. The lignin content in harpin-treated tobacco leaves was about 2.5-fold as compared with the controls 24 h after inoculation. There were six isozymes of POD (pI 9.5, pI 8.7, pI 5.3, pI 4.4, pI 3.7, and pI 3.5) and seven isozymes of laccase (pI 9.4, pI 8.6, pI 7.8, pI 5.4, pI 4.5, pI 3.8, and pI 3.6) identified by isoelectric point in extracts of harpin-inoculated tobacco leaves. POD isozymes (pI 4.4, pI 5.3 and pI 8.7) and laccase isozyme (pI 7.8) only appeared in harpin-inoculated tissues. The increased POD isozymes (pI 4.4, pI 8.7, pI 9.5) are correlated with the rise of transcripts of these enzymes confirmed by the method of reverse transcriptase-polymerase chain reaction (RT-PCR).

Plants defence responses are often associated with the development of the so-called hypersensitive response (HR), a form of PCD that confines the pathogen to the infection site. The sharp boundary of the HR suggests the existence of efficient mechanisms that control cell death and survival. The Arabidopsis transcription factor AtMYB30 positively regulates plant defence and HR responses by enhancing the synthesis of sphingolipid-containing Very Long Chain Fatty Acids (VLCFA) after bacterial infection. The activity of AtMYB30 is tightly controlled inside plant cells through protein-protein interactions and post-translational modifications. During my PhD, we identified a protease of the subtilase family (AtSBT5.2) as a AtMYB30-interacting partner. Interestingly, we have shown that the AtSBT5.2 transcript is alternatively spliced, leading to the production of two distinct gene products that encode either a secreted [AtSBT5.2(a)] or an intracellular [AtSBT5.2(b)] protein. The specific interaction between AtMYB30 and AtSBT5.2(b), but not AtSBT5.2(a), leads to AtMYB30 specific retention outside of the nucleus in small intracellular vesicles. atsbt5.2 Arabidopsis mutant plants, in which both AtSBT5.2(a) and AtSBT5.2(b) expression was abolished, displayed enhanced HR and defence responses. The fact that this phenotype is abolished in an atmyb30 mutant background suggests that AtSBT5.2 is a negative regulator of AtMYB30-mediated disease resistance. Importantly, overexpression of the AtSBT5.2(b), but not the AtSBT5.2(a), isoform in the atsbt5.2 mutant background reverts the phenotypes displayed by atsbt5.2 mutant plants, suggesting that AtSBT5.2(b) specifically represses AtMYB30-mediated defence.

No description available.

Infection of the resistant Arabidopsis ecotype Di-17 with Turnip Crinkle Virus (TCV) elicits hypersensitive response (HR), accompanied by increased expression of defense genes. HR to TCV is conferred by HRT, which encodes a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of resistance (R) protein. In contrast to HR, resistance requires HRT and a recessive locus designated rrt. Unlike most CC-NBS-LRR R proteins, HRT-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also dependent on salicylic acid (SA) pathway and light. A dark treatment, immediately following TCV inoculation, suppresses HR, resistance and activation of a majority of the TCV-induced genes. To determine the genetic, molecular and biochemical basis of light-dependent defense pathway, we studied the role of various photoreceptors in HRT-mediated resistance to TCV, HRT protein levels and its localization. Interestingly, mutation in blue-light photoreceptors led to degradation of HRT via a proteasome-dependent pathway and resulted in susceptibility to TCV. Exogenous application of SA induced transcription of HRT, which restored HRT levels in some, but not all, mutant backgrounds. These results show that different photoreceptors function distinctly in maintaining post-transcriptional stability of HRT. In addition to photoreceptors, HRT also forms a complex with several other proteins, many of which participate in the RNA silencing pathway and are required for HRT-mediated resistance. Together, our results suggest that HRT forms a multi-protein complex and that HRT-mediated signaling involves reconstitution of this complex.

Turnip crinkle virus (TCV) has been implicated in the suppression of the hypersensitive response (HR), a type of programmed cell death induced during active resistance in Arabidopsis thaliana. In order to investigate the involvement of individual viral components in mediating suppression, TCV genes were cloned for use in an Agrobacterium tumefaciens mediated transient expression in Nicotiana benthamiana. Agroinfiltration of the HR-inducing avrPto/Pto system in conjunction with individual TCV genes has identified the p38 gene, which encodes the viral coat protein, as the gene responsible for the cell death suppression phenotype. The extent of cell death suppression by coat protein was quantified and found to be equal to the level of suppression by the whole virus and AvrPtoB, another cell death inhibitor from bacteria. Thus, the coat protein alone is sufficient to inhibit the HR in plants. Further, the effect of TCV on HR initiation by an avirulence factor from an unrelated bacterial pathogen was investigated. The presence of TCV does not affect the production, secretion or cellular processing of the bacterial avirulence factor.

<p>  </p> <p>Forward genetics discovery of the molecular basis of induced mutants has fundamentally contributed to our understanding of basic biological processes such as metabolism, cell dynamics, growth, and development. Advances in Next-Generation Sequencing (NGS) technologies enabled rapid genome sequencing but also come with limitations such as sequencing errors, dependence on reference genome accuracy, and alignment errors. By incorporating pedigree information to help correct for some errors I optimized variant calling and filtering strategies to respond to experimental design. This led to the identification of multiple causative alleles, the detection of pedigree errors, and an ability to explore the mutational spectrum of multiple mutagens in Arabidopsis. Similar to the problems in forward genetic discovery of mutant alleles, variation in genomes complicates the analysis of gene expression affected by natural variation. The plant hypersensitive response (HR) is a highly localized and rapid form of programmed cell death that plants use to contain biotrophic pathogens. Substantial natural variation exists in the mechanisms that trigger and control HR, yet a complete understanding of the molecular mechanisms modulating HR is lacking. I explored the gene expression consequences of the plant HR in maize using a semi-dominant mutant encoding a constitutively active HR-inducing Nucleotide Binding Site Leucine Rich Repeat protein, <em>Rp1-D21,</em> derived from the receptor responsible for perceiving certain strains of the common rust <em>Puccinia sorghi</em>. Differentially expressed genes (DEG) in response to <em>Rp1-D21</em> were identified in different genetic backgrounds and hybrids that exhibit divergent enhancing (NC350) or suppressing (H95, B73) effects on the visual manifestations of HR. To enable this analysis, I created anonymized reference genomes for each comparison, so that the reference genome induced less bias in the mapping steps. Comprehensive identification of DEG corroborated the visual phenotypes and provided the identities of genes influential in plant hypersensitive response for further studies. The locations of expression quantitative trait loci (eQTL) that determined the differential response of NC350 and B73 were identified using 198 F1 families generated by crossing B73 x NC350 RIL population and <em>Rp1-D21</em>/+ in H95. This identified 3514 eQTL controlling the variability in differential expression between mutant versus wild-type. <em>Trans-</em>eQTL were dramatically arranged in the genome and identified 17 hotspots with more than 200 genes influenced by each locus. A single locus significantly affected expression variation in 5700 genes, 5396 (94.7%) of which were DGE. An allele specific expression analysis of NC350 x H95 and B73 x H95 F1 hybrids with and without <em>Rp1-D21</em> identified <em>cis-</em>eQTL and ASE at a subset of these genes. Bias in the confirmation of eQTL by ASE was still present despite the anonymized reference genomes indicating that additional efforts to improve signal processing in these experiments is needed.</p>