Nonhost Disease Resistance in Pea: Chitosan's Suggested Role in DNA Minor Groove Actions Relative to Phytoalexin-Eliciting Anti-Cancer Compounds.

A stable intense resistance called "nonhost resistance" generates a complete multiple-gene resistance against plant pathogenic species that are not pathogens of pea such as the bean pathogen, Fusarium solani f. sp. phaseoli (Fsph). Chitosan is a natural nonhost resistance response gene activator of defense responses in peas. Chitosan may share with cancer-treatment compounds, netropsin and some anti-cancer drugs, a DNA minor groove target in plant host tissue. The chitosan heptamer and netropsin have the appropriate size and charge to reside in the DNA minor groove. The localization of a percentage of administered radio-labeled chitosan in the nucleus of plant tissue in vivo indicates its potential to transport to site(s) within the nuclear chromatin (1,2). Other minor groove-localizing compounds administered to pea tissue activate the same secondary plant pathway that terminates in the production of the anti-fungal isoflavonoid, pisatin an indicator of the generated resistance response. Some DNA minor groove compounds also induce defense genes designated as "pathogenesis-related" (PR) genes. Hypothetically, DNA targeting components alter host DNA in a manner enabling the transcription of defense genes previously silenced or minimally expressed. Defense-response-elicitors can directly (a) target host DNA at the site of transcription or (b) act by a series of cascading events beginning at the cell membrane and indirectly influence transcription. A single defense response, pisatin induction, induced by chitosan and compounds with known DNA minor groove attachment potential was followed herein. A hypothesis is formulated suggesting that this DNA target may be accountable for a portion of the defense response generated in nonhost resistance.

DNA Damage and Chromatin Conformation Changes Confer Nonhost Resistance: A Hypothesis Based on Effects of Anti-cancer Agents on Plant Defense Responses.

Over the last decades, medical research has utilized DNA altering procedures in cancer treatments with the objective of killing cells or suppressing cell proliferation. Simultaneous research related to enhancing disease resistance in plants reported that alterations in DNA can enhance defense responses. These two opposite perspectives have in common their effects on the center for gene transcription, the nuclear chromatin. A review of selected research from both anticancer- and plant defense-related research provides examples of some specific DNA altering actions: DNA helical distortion, DNA intercalation, DNA base substitution, DNA single cleavage by DNases, DNA alkylation/methylation, and DNA binding/exclusion. The actions of the pertinent agents are compared, and their proposed modes of action are described in this study. Many of the DNA specific agents affecting resistance responses in plants, e.g., the model system using pea endocarp tissue, are indeed anticancer agents. The tumor cell death or growth suppression in cancer cells following high level treatments may be accompanied with chromatin distortions. Likewise, in plants, DNA-specific agents activate enhanced expression of many genes including defense genes, probably due to the chromatin alterations resulting from the agents. Here, we propose a hypothesis that DNA damage and chromatin structural changes are central mechanisms in initiating defense gene transcription during the nonhost resistance response in plants.

Non-host Resistance: DNA Damage Is Associated with SA Signaling for Induction of PR Genes and Contributes to the Growth Suppression of a Pea Pathogen on Pea Endocarp Tissue.

Salicylic acid (SA) has been reported to induce plant defense responses. The transcriptions of defense genes that are responsible for a given plant's resistance to an array of plant pathogens are activated in a process called non-host resistance. Biotic signals capable of carrying out the activation of pathogenesis-related (PR) genes in pea tissue include fungal DNase and chitosan, two components released from Fusarium solani spores that are known to target host DNA. Recent reports indicate that SA also has a physical affinity for DNA. Here, we report that SA-induced reactive oxygen species release results in fragment alterations in pea nuclear DNA and cytologically detectable diameter and structural changes in the pea host nuclei. Additionally, we examine the subsequent SA-related increase of resistance to the true pea pathogen F. solani f.sp. pisi and the accumulation of the phytoalexin pisatin. This is the first report showing that SA-induced PR gene activation may be attributed to the host pea genomic DNA damage and that at certain concentrations, SA can be temporally associated with subsequent increases in the defense response of this legume.

A Simple and Rapid Assay for Measuring Phytoalexin Pisatin, an Indicator of Plant Defense Response in Pea (Pisum sativum L.).

Phytoalexins are antimicrobial substance synthesized in plants upon pathogen infection. Pisatin (Pisum sativum phytoalexin) is the major phytoalexin in pea, while it is also a valuable indicator of plant defense response. Pisatin can be quantitated in various methods from classical organic chemistry to Mass-spectrometry analysis. Here we describe a procedure with high reproducibility and simplicity that can easily handle large numbers of treatments. The method only requires a spectrophotometer as laboratory equipment, does not require any special analytical instruments (e.g., HPLC, mass spectrometers, etc.) to measure the phytoalexin molecule quantitatively, i.e., most scientific laboratories can perform the experiment.

Anatomy of a nonhost disease resistance response of pea to Fusarium solani: PR gene elicitation via DNase, chitosan and chromatin alterations.

Of the multiplicity of plant pathogens in nature, only a few are virulent on a given plant species. Conversely, plants develop a rapid "nonhost" resistance response to the majority of the pathogens. The anatomy of the nonhost resistance of pea endocarp tissue against a pathogen of bean, Fusarium solani f.sp. phaseoli (Fsph) and the susceptibility of pea to F. solani f sp. pisi (Fspi) has been described cytologically, biochemically and molecular-biologically. Cytological changes have been followed by electron microscope and stain differentiation under white and UV light. The induction of changes in transcription, protein synthesis, expression of pathogenesis-related (PR) genes, and increases in metabolic pathways culminating in low molecular weight, antifungal compounds are described biochemically. Molecular changes initiated by fungal signals to host organelles, primarily to chromatin within host nuclei, are identified according to source of the signal and the mechanisms utilized in activating defense genes. The functions of some PR genes are defined. A hypothesis based on this data is developed to explain both why fungal growth is suppressed in nonhost resistance and why growth can continue in a susceptible reaction.

EDTA a novel inducer of pisatin, a phytoalexin indicator of the non-host resistance in peas.

Pea pod endocarp suppresses the growth of an inappropriate fungus or non-pathogen by generating a "non-host resistance response" that completely suppresses growth of the challenging fungus within 6 h. Most of the components of this resistance response including pisatin production can be elicited by an extensive number of both biotic and abiotic inducers. Thus this phytoalexin serves as an indicator to be used in evaluating the chemical properties of inducers that can initiate the resistance response. Many of the pisatin inducers are reported to interact with DNA and potentially cause DNA damage. Here we propose that EDTA (ethylenediaminetetraacetic acid) is an elicitor to evoke non-host resistance in plants. EDTA is manufactured as a chelating agent, however at low concentration it is a strong elicitor, inducing the phytoalexin pisatin, cellular DNA damage and defense-responsive genes. It is capable of activating complete resistance in peas against a pea pathogen. Since there is also an accompanying fragmentation of pea DNA and alteration in the size of pea nuclei, the potential biochemical insult as a metal chelator may not be its primary action. The potential effects of EDTA on the structure of DNA within pea chromatin may assist the transcription of plant defense genes.

Multiple effects of chitosan on plant systems: solid science or hype.

Chitosan, a naturally occurring polymer, became available in the 1980s in industrial quantities enabling it to be tested as an agricultural chemical. A usual procedure for developing agricultural chemicals starts by testing a number of different chemically synthesized molecules on a targeted biological system. Alternately, chitosan has been investigated as a single natural molecule assayed with numerous biological systems. This report describes the unique properties of the molecule and its oligomers, primarily in plant defense, additionally in yield increase, induction of cell death and stomatal closing. The plant plasma membrane and nuclear chromatin have been proposed as targets, though chitosan oligomers enter most regions of the cell. Subsequent changes occur in: cell membranes, chromatin, DNA, calcium, MAP kinase, oxidative burst, reactive oxygen species (ROS), callose, pathogenesis related (PR) genes/proteins, and phytoalexins. Chitosan oligomer mode(s) of action are proposed for different plant systems. Chitosan efficacy was based on documentation from published data. Attention was given to how chitosan, either applied externally or released by fungal inoculum, is transferred into plant cells and its subsequent action upon membrane and/or chromatin components. Within is a proposed scheme describing chitosan generation, signaling routes and mechanisms of defense gene activation. Examples of beneficial chitosan applications to major crop/food plants were included.

Nuclease released by Verticillium dahliae is a signal for non-host resistance.

A DNase released from the fungal pathogen of bean, Fusarium solani f. sp. phaseoli (Fsph), was previously shown to signal the activation of total disease resistance and activate pathogenesis-related (PR) genes in pea. Data in the current study which used the pea-endocarp model to research non-host resistance, indicated that DNase released by Verticillium dahliae (Vd), pathogenic on potato also has non-host resistance-inducing capabilities in peas. Other strains of Vd that release DNase are pathogenic on other plant species. DNase catalytic activity was also released from representative genera of other pathogenic fungi. Purified VdDNase induced pisatin and pea gene DRR49 (PR-10 gene) in pea endocarp tissue. VdDNase reduced the in vitro growth of Vd but completely inhibited that of F. solani f. sp. pisi (Fspi) and a Colletotrichum pathogen of potato. VdDNase (2 units) applied to pea endocarp tissue both broke resistance to Fsph and increased resistance to Fspi. Pea DNA damage generated both by the VdDNase enzyme and the intact Vd spores indicated that the host DNA alteration is a component of the non-host resistance response (innate immunity). These data support the previously reported inductive potential of fungal DNase and further implicate fungal DNases as signals in activating non-host resistance responses.

Fungal mitochondrial DNases: effectors with the potential to activate plant defenses in nonhost resistance.

Previous reports on the model nonhost resistance interaction between Fusarium solani f. sp. phaseoli and pea endocarp tissue have described the disease resistance-signaling role of a fungal DNase1-like protein. The response resulted in no further growth beyond spore germination. This F. solani f. sp. phaseoli DNase gene, constructed with a pathogenesis-related (PR) gene promoter, when transferred to tobacco, generated resistance against Pseudomonas syringe pv. tabaci. The current analytical/theoretical article proposes similar roles for the additional nuclear and mitochondrial nucleases, the coding regions for which are identified in newly available fungal genome sequences. The amino acid sequence homologies within functional domains are conserved within a wide array of fungi. The potato pathogen Verticillium dahliae nuclease was divergent from that of the saprophyte, yeast; however, the purified DNase from yeast also elicited nonhost defense responses in pea, including pisatin accumulation, PR gene induction, and resistance against a true pea pathogen. The yeast mitochondrial DNase gene (open reading frame) predictably codes for a signal peptide providing the mechanism for secretion. Mitochondrial DNase genes appear to provide an unlimited source of components for developing transgenic resistance in all transformable plants.

Pea-Fusarium solani interactions contributions of a system toward understanding disease resistance.

This mini-review points to the usefulness of the pea-Fusarium solani interaction in researching the biochemical and molecular aspects of the nonhost resistance components of peas. This interaction has been researched to evaluate the resistance roles of the phytoalexin, pisatin, the cuticle barrier, and the activation of the nonhost resistance response. Concurrently, evaluations of associated signaling processes and the tools possessed by the pathogen to contend with host obstacles were included. The properties of some pathogenesis-related genes of pea and their regulation and contribution to resistance are discussed. A proposed action of two biotic elicitors on both chromatin conformation and the architectural transcription factor, HMG A, is presented and includes time lines of events within the host immune response.

A Promoter from Pea Gene DRR206 Is Suitable to Regulate an Elicitor-Coding Gene and Develop Disease Resistance.

ABSTRACT Plant nonhost disease resistance is characterized by the induction of multiple defense genes. The pea DRR206 gene is induced following inoculation with pathogens and treatment with abiotic agents, and moderately induced by wounding. A deletion series of DRR206 promoter segments was fused with the beta-glucuronidase (GUS) reporter gene and transiently transferred to tobacco, potato, and pea. GUS activity revealed that two upstream regions of the DRR206 promoter were particularly important for activation in the three plant species. Putative cis regulatory elements within the DRR206 promoter included a wound/pathogen- inducible box (W/P-box) and a WRKY box (W-box). Gel shift assays with nuclear extracts from treated and untreated tissue with the W/P-box revealed both similar and unique protein-DNA complexes from pea, potato, and tobacco. Tobacco was stably transformed with gene constructs of the DRR206 promoter fused with a DNase elicitor gene from Fusarium solani f. sp. phaseoli, FsphDNase. Pathogenicity tests indicated that the FsphDNase elicitor conferred resistance against Pseudomonas syringae pv. tabaci and Alternaria alternata in tobacco. Transgenic potatoes showed some sensitivity to the FsphDNase gene providing less protection against Phytophthora infestans. Thus, the elicitor-coding gene, FsphDNase, is capable of generating resistance in a heterologous plant system (tobacco) when fused with defined regions of the pea DRR206 promoter.

Analysis of pea HMG-I/Y expression suggests a role in defence gene regulation.

SUMMARY HMG-I/Y proteins are characterized by the presence of AT-hook motifs, DNA binding domains that recognize AT-rich tracts of DNA. By facilitating protein:protein and protein:DNA interactions in the vicinity of these AT-rich binding sites, HMG-I/Y positively or negatively regulates gene expression. Several pea defence gene promoters have AT-rich tracts of DNA that are potential targets for modulation via HMG-I/Y. In this study, a comparison of the expression of a pea defence gene (DRR206) mRNA relative to the expression of HMG-I/Y mRNA was monitored by Northern analysis following the inoculation of a fungal pathogen, Fusarium solani or treatment with chitosan and a F. solani DNase (Fsph DNase). In pea pod endocarp tissue, HMG-I/Y expression was observed at high levels in untreated tissue and at lower levels 6 h following inoculation or wounding of the tissue. Western blots with an antipea HMG-I/Y polyclonal antibody also revealed that pea HMG-I/Y is expressed at decreased levels 6 h following inoculation or elicitor treatment. HMG-I/Y extracted from pea caused alterations in the gel migration of radio-labelled AT-rich sequences from the pea DRR206 promoter, suggesting that similar interactions could exist in vivo. Agroinfiltration was utilized to express the pea HMG-I/Y gene in tobacco containing a chimeric gene fusion of a promoter from the PR gene, DRR206, and the beta-glucuronidase (GUS) reporter gene. Transient expression of pea HMG-I/Y led to a decrease in GUS reporter gene activity in the heterologous tobacco system. These data implicate pea HMG-I/Y abundance in the down-regulation of DRR206 gene expression, and possibly HMG-I/Y depletion in the expression of defence genes in pea.