| China is by far the largest producer，in terms of the cultivation scale andproduction volume of Saccharina japonica. It accounted for60%of the totalproduction of the world. The artificially intensively cultivated S. japonica might favordisease outbreaks, which regularly lead to the destructive loss of25-30%of thevolume at a regional scale. Therefore, the purpose of gaining a profoundunderstanding of pathogenic bacteria and virulence as well as the defense responsemechanisms of S. japonica not only facilitating the early prevention and diagnosis ofthe disease outbreaks, but also formulating the reasonable production managementmeasures for reducing and mitigating the incidence of the disease.According to our previous study, flg22which is an effective elicitor in higherplants could cause a series of immune defense responses in S. japonica. Our findingsindicate that flg22is also an effective elicitor for S. japonica. The question proposedhere is that whether flg22-derived peptides can also trigger defense responses in S.japonica? Therefore, in this study, we investigated the elicitor activity of flg22and itsderived peptides, the minimum epitope of flagellin as well as the active site of flg22in the female gametophytes of S. japonica to establish stable elicitor-S. japonicaexperimental models. this model had achieved a preliminary work foundation of notonly identification of pathogen or elicitor receptor, but also the signal transductionpathways. Our results would enrich the theory of plant molecular pathology and helpto develop the new S. japonica varieties with disease resistance. The results are asfollows:1, Quantitative detection of H2O2in the female gametophytes of S. japonicainduced by flg22and its derived peptides.Using luminol fluorescence detection method, the production of H2O2was investigated induced by the different concentrations of flg22and its derived peptidesof flg15、flg14及flg22D43A in the female gametophytes of S. japonica. Within the rangeof0-200μM, the production of H2O2increased with the increase of the peptideconcentrations in flg22-and flg15-induced female gametophytes. When thepeptide concentrations up to260μM, there was no obvious rise in the release of H2O2in the female gametophytes. Moreover, it was required that concentration of flg22wasslightly lower than that of flg15for the same production of H2O2.Our resultsindicated that flg22had higher elicitor activity that flg15and there was no significantdifferences of elicitor activity between flg22and flg15. Regarding to flg14, theproduction of H2O2increased gradually with the increment of flg14concentrationsduring the range of400to1000μM.Because no H2O2was detected in the female gametophytes induced byflg22D43A, it was implied that aspartic acid (D) at position43from the N-terminusof a flagellin also played a crucial role when flg22acting effective elicitor for S.japonica.Quantity detection of H2O2was studied at different inducing time course of byflg22, flg15, flg14and flg22D43A at the concentration of90μM. Our results showedthat the production of H2O2induced by flg22and flg15increased with the inducingtime course within0-2h and reached a peak at2h about2400RLU and1865RLU(relative light unit), respectively. After2h, the production of increased over theextension of time declined gradually with the inducing time course. The production ofH2O2returned to the initial level after6h. At the same inducing time course, theproduction of H2O2induced by flg22was slightly higher than that induced by flg15.The production of H2O2induced by flg14and flg22D43A had no obvious changewithin6h.2, Histological detection of ROS in the female gametophytes of S. japonicainduced by flg22and its derived peptides.Using DCFH-DA fluorescent probe method, histological detection of ROS in thefemale gametophytes of S. japonica induced by flg22and flg22-derived peptides wereobserved under the fluorescence microscope. Our results showed that weak green DCF fluorescence was recorded in the female gametophytes induced by flg22andflg15at the concentrations of20μM. Fluorescent intensity increased with theconcentrations of both flg22and flg15in the range of20-200μM. The greenfluorescence was observed in flg14induced female gametophytes with theconcentration of600μM. Fluorescence intensity increased with the increase ofconcentration of600-1000μM. For flg22D43A, no ROS was detected in the femalegametophytes even at a concentration of1000μM. The histological observationshowed consistent results with the results of luminol fluorescence detection.3, Growth inhibition of flg22and flg22-derived peptides on the femalegametophytes of S. japonicaGrowth inhibition of flg22, flg15, flg14and flg22D43A on female gametophytesof S. japonica was identified at the respective concentration of1μM after beingco-cultured for40d. Our results showed that the fresh weights of flg22-and flg15-induced S. japonica were0.14g and0.2g respectively, which were less than a halffresh weight of the control with0.45g. Moreover, flg15had weaker inhibitory effecton the growth of female gametophytes compared to flg22. Flg14showed a little bitgrowth inhibition with0.35g fresh weight. While, flg22D43A had no inhibitoryeffects on the growth of S. japonica female gametophytes.4, The quantitative detection of H2O2in the female gametophytes of S. japonicainduced by flg15and flg15-derived peptides.Elicitor activity of flg15and flg15-derived peptides flg15-Δ1, flg15-Δ2, flg15-Δ3,flg15-Δ4, flg15-Δ5, flg15-Δ6, flg15-Δ7and flg15-Δ8, which were truncated from theC-terminus amino acid of flg15, were tested. Using luminol fluorescence detectionmethod, according to H2O2released from the induced female gametophytes by flg15,flg15-Δ1, flg15-Δ2, flg15-Δ3, flg15-Δ4, flg15-Δ5, flg15-Δ6, flg15-Δ7and flg15-Δ8,we found that the production of H2O2increased with the increase of concentrations offlg15-Δ1, flg15-Δ2and flg15-Δ3in the range of200-1000μM. Finally, theproduction of H2O2reached the same level as what released from flg15inducedfemale gametophytes with8000RLU. There was no significant differences in elicitoractivity among flg15-Δ1, flg15-Δ2and flg15-Δ3. The production of H2O2induced by flg15-Δ4had a similar trend to those of flg15-Δ1, flg15-Δ2and flg15-Δ3withinconcentrations of200-1000μM with lower elicitor activity compared to those offlg15-Δ1, flg15-Δ2and flg15-Δ3. No production of H2O2was detected in the inducedfemale gametophytes by flg15-Δ5, flg15-Δ6, flg15-Δ7and flg15-Δ8. This findingsindicated that were not the effective elicitors for S. japonica. Our results suggestedthat the elicitor activity of all flg15-derived peptides declined and truncated5amminoacids from the C-terminus of flg15resulted in the disappearance of elicitor activity.According to our data, flg15is an effective elicitor which could induce defenseresponses and is also the minimum flagellin epitope for S. japonica. Similar to thosehigher plants, aspartic acid (D) at position43from the N-terminus of a flagellin play acrucial role for elicitor activity of flg22in S. japonica. |
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