Salicylic Acid Biosynthesis And Associated Mechanisms Towards Responding Fusarium Graminearum In Barley

Salicylic acid?SA?plays an important role in signal transduction and disease resistance.In model plant Arabidopsis,SA can be synthesized by two enzymatic pathways:1)isochorismate synthase?ICS?and 2)phenylalanine ammonia lyase?PAL?.However,the SA biosynthesis pathway remains largely unknown in Triticeae.In the current study,we cloned the HvICS and seven HvPAL genes from barley and generated their overexpression?OE?and RNA interference?RNAi?barley plants.We further determined the SA content in transgenic barley,and challenged the transgenic plants using Fusarium graminearum.The current study elucidated the SA siosynthesis in barley and illustrate its effect on disease resistance.The results are as follows:1.Sequence and expression of target genes:In barley,HvICS is a single copy gene that sits on the chromosome 5H,but there are at least 10 PAL genes onchromosomes 1H,2H,and6H.The ICS proteins of barley,rice and Brachypodium shared<51%identity when compared with the ICS protein of Arabidopsis.Despite the considerable amino acid differece,the predicted 3-D structure of various ICS proteins were comparable.On the other hand,all PAL proteins are featured by the Ala-Ser-Gly?ASG?signature on their activation site,which indirectly suggests the PAL proteins use similar mechanism for function.In barley‘Golden Promise',HvICS dispiayed ubiquitous expression in leaves,stem and sheath,but relatively low expression in spikes.However,the HvPALs genes showed an equally biased organ-specific pattern of expression.2.HvICS and/or HvPAL contributes to the SA biosynthesis in barley:We prepared OE and RNAi constructs for HvICS and HvPAL genes,which were used to transform barley‘Golden Promise'.The barley transformation rate is 5.3%as revealed by the number of transgenic events per 100 calli infected.There was no significant difference between HvPAL T₁ transgenic plants and wild-type?WT?plants in plant height,tiller number,flowering time and resistance to F.graminearum.In contrast,for the HvICS RNAi construct,the callus proliferation and plantlet regeneration were significantly inhibited during the tissue culture steps that resulted in the failaure of transformation.By supplying 100?M SA in media,we were able to rescue three transgenic plants,which displayed irregular phenotypes:dwarf,multiple tillers,late flowering and susceptible to F.graminearum.In RNAi plants,the HvICS mRNA was reduced to 49%to 73%of those in the WT control,but the SA content was comparable between the RNAi plants and the WT control.In OE plants,the HvICS mRNA increased about 16-to 18-fold as compared to those in the WT control,but the SA content only increased about 1.6-fold.These data indicated that the HvICS gene plays role in the SA biosynthesisin barley and impacts physiological responses.3.HvICS affects the F.graminearum resistance in barley:We used transgenic plants to test the F.graminearum resistance in leaves and spikes.The HvICS RNAi plants had larger lesions of F.graminearum,about 2-fold bigger than that of the WT control.The HvICS mRNA levels of the OE and WT plants were significantly upregulated by the F.graminearum,approximately 2-fold of those in plants treated by water?H₂O?.The WT plants,when challenged by the F.graminearum,also produced 19 times of SAthan those inWT plants treated by water.In contrast,the HvICS RNAi plant had a low level of the HvICS mRNA and the SA content regardless of the F.graminearum infection.The ICS OE spikes were relatively resistant toF.graminearum at 24 hours after innoclation?hai?and 48hai.Therefore,the ICS gene could affect the resistance to F.graminearum by modulating the accumulation of SA,H₂O₂,O₂^-,and reactive-oxygen associated enzymatic activities.4.Barley shows no systemic acquired resistance?SAR?to F.graminearum:A double inoculation procedure?‘inducer'followed by‘challenger'?were used for the SAR test in the WT barley.The result showed the infection of the inducer on one leaf did not induce SAR against the challenger on a different leaf.In addition,there was no significant change in the ICS expression and the SA content at uninfected sites.Therefore,in barley,there was no practical SAR for preventing the secondery infection,at least for F.graminearum tested here.5.HvICS and SA regulate plant growth and development:Different levels of SA were supplied during barley germination.Low levels of SA?50-100?M?promoted shoot and root growth,however,high concentration of SA?200-500?M?inhibited the shoot and root growth.During transgenic research,callus proliferation and plantlet regeneration were arrested when dealing with the HvICS RNAi construct,but the supplement of 100?M SApartially rescued HvICS RNAi transgenic cells.In addition,the mutation of the ICS-A locus lead to dwarfism,yellowing and late flowring in the durum wheat‘Kronos'.