Evaluation of the role of PGIPs in plant defense responses

[摘要] ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. Theevents that prepare the plant for, and follow plant-pathogenic interactions, areextremely complex and have been the topic of intensive investigation in recentyears. These interactions involve a plethora of genes and proteins, and intricateregulation thereof; from the host and pathogen alike. Studying the contribution ofsingle genes and their encoded proteins to the molecular dialogue between plantand pathogen has been a focus of plant molecular biologists.To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP)was recently cloned from Vitis vinifera. These proteins have the ability to inhibitfungal endopolygalacturonases (ePGs), enzymes which have been shown to berequired for the full virulence of several fungi on their respective plant hosts. Theactivity of PGIP in inhibiting fungal macerating enzymes is particularly attractivefor the improvement of disease tolerance of crop species. The VvPGIP-encodinggene was subsequently transferred to Nicotiana tabacum for high-level expressionof VvPGIP. These transgenic plants were found to be less susceptible to infectionby Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePGinhibition by protein extracts from these lines correlated to the observed decreasein susceptibility to B. cinerea. This study expands on previous findings bycorroborating the antifungal nature of the introduced PGIP by whole-plant, timecourseinfection assays. Six transgenic tobacco lines and an untransformed wildtype(WT) were infected and the lesions measured daily from day three to seven,and again at day 15. The transgenic lines exhibited smaller lesions sizes fromthree to seven days post-inoculation, although these differences only becamestatistically significant following seven days of incubation. At this point, four of thesix lines exhibited significantly smaller lesions than the WT, with reductions indisease susceptibility ranging between 46 and 69% as compared to the WT. Twoof the lines exhibited disease susceptibility comparable to the WT. In theseresistant plant lines, a correlation could be drawn between Vvpgip1 expression,PGIP activity and ePG inhibition. These lines were therefore considered to bePGIP-specific resistant lines, and provided ideal resources to further study thepossible in planta roles of PGIP in plant defense.The current hypothesis regarding the role(s) of PGIP in plant defense is twofold.Firstly, PGIPs have the ability to specifically and effectively inhibit fungalePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively,unhindered action of these enzymes results in maceration of plant tissue andultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, theinhibition of ePGs prolongs the existence of oligogalacturonides, molecules withthe ability to activate plant defense responses. Thus, PGIPs limit tissue damageby inhibition of ePG; this inhibition results in activation of plant defense responsesaimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenicplant lines overexpressing PGIP-encoding genes. However, none of thesepublications could expand on the current hypotheses regarding the possible inplanta roles of PGIP in plant defense. In this study we used transgenic tobaccolines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP.Transcriptomic and hormonal analyses were performed on these lines and a WTline, both before and following inoculation with Botrytis cinerea.Transcriptomic analysis was performed on uninfected as well as infectedtobacco leaf material utilizing a Solanum tuberosum microarray. From the analysiswith healthy, uninfected plant material, it became clear that genes involved in cellwall metabolism were differentially expressed between the transgenic lines andthe WT. Under these conditions, it could be shown and confirmed that the geneencoding tobacco xyloglucan endotransglycosylase (XET/XTH) wasdownregulated in the transgenic lines. Additionally, genes involved in the ligninbiosynthetic pathway were affected in the individual transgenic lines. Biochemicalevidence corroborated the indication of increased lignin deposition in their cellwalls. Additionally, phytohormone profiling revealed an increased indole-aceticacid content in the transgenic lines. These results show that constitutive levels ofPGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which mayhave a positive impact on the observed reduced susceptibilities of these plants.An additional role for PGIP in the contribution to plant defenses is thereforeproposed. PGIP may directly influence defense responses in the plant leading tothe strengthening of cell walls. This might occur by virtue of its structural featuresor its integration in the cell wall. These reinforced cell walls are thus 'primedbefore pathogen ingress and contribute to the decrease in disease susceptibilityobserved in lines accumulating high levels of PGIP.Transcriptional and hormonal analyses, at the localized response, wereperformed on Botrytis-infected leaf tissue of the transgenic lines and a WT line.Several Botrytis responsive genes were found to be upregulated in both the WTand the transgenic lines. Although limited differential expression was observedbetween the two genotypes, the analyses identified a gene which wasupregulated two-fold in the transgenic lines, as compared to WT. This wasconfirmed by quantitative Real-Time PCR. This gene is involved in thelipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis ofthe divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitoryeffects on Botrytis spore germination. Phytohormone profiling revealed that thetransgenic lines accumulated more of the defense-related hormone pool ofjasmonates. These are formed via the 13-LOX pathway and have been shown tobe important for the restriction of Botrytis growth at the site of infection.Collectively, the results from the infection analyses indicate that in thesetransgenic lines, both branches of the lipoxygenase pathway are differentiallyinduced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acidcould be observed, although this hormone did not accumulate to significantlyhigher levels. These results are the first report of differential induction of adefense-related pathway in pgip-overexpressing lines and substantiate theproposal that following ePG inhibition by PGIP, signaling which activates plantdefense responses, takes place.Taken together, these results significantly contribute to our understanding ofthe in planta role of PGIP in plant defense responses.

[发布机构] Stellenbosch University