Effects Of Infection By Leifsonia Xyli Subsp.xyli On The Tissue Structure And Key Enzymes Activity Of C4 Pathway In Sugarcane

Ratoon stuning disease?RSD?is a bacterial disease that has plagued the sugarcane planting countries and regions in the world for a long time.The pathogen Leifsonia xyli subsp.xyli?Lxx?is a fastidious gram-positive positive bacteria,and it is difficult to culture in vitro,therefore,the research progress on the pathogenic mechanism of interaction between Lxx and sugarcane as a C4 crop has been slow.Until recently,it was discovered that GFP-labeled Lxx appeared in vascular bundle sheath cells and mesophyll cells in the“kranz anatomy”of sugarcane,indicating that Lxx was not a xylem-dependent strain that researchers had always thought,and opened up new possibilities for the pathogenicity of Lxx.The specialized“kranz anatomy”of C4 crop is closely related to the C4 cycle in the photosynthetic carbon fixation pathway.Whether Lxx-infection causes damage to the structure in sugarcane and affects the C4 cycle and function of photosynthetic carbon fixation of the sugarcane needs to be explored.The aim of this study was to investigate the effects of Lxx-infection on the C4 cycle in photosynthetic carbon fixation pathways of sugarcane from the cellular,physiological and molecular levels,to explore the pathogenic mechanism of Lxx in sugarcane.The effects of Lxx-infection on the“kranz anatomy”of sugarcane were analyzed with a healthy and the obviously dwarfed RSD plants,and the variety was Badila.Meanwhile,starch,the final product of photosynthetic carbon fixation in this region,was dyed and in situ PCR was used to investigate the colonization niche of Lxx.The healthy seed-stems was used as a test material for Lxx-inoculation study,which based on the separation,culture and identification of Lxx.Then the quantitative analysis of Lxx in leaves was conducted by qPCR,to investigate the rapid accumulation period of Lxx in leaves,and transcriptome sequencing was performed on the leaves of RSD-infected and control group during this period,the differential expression of related genes in the C4 cycle pathway was analyzed,and the key enzymes activity and gene expression changes of the C4 pathway in the process of Lxx-infection were measured.The main results are as follows:?1?Compared to healthy sugarcane plants,the gaps among the vascular bundle sheath cells in the inside of the“kranz anatomy”in RSD sugarcane leaves increased,the boundary of the lateral vascular bundle sheath cells was blurred and the inclusions were dispersed,and the cells in the xylem,phloem and the procambia of the vascular organization in the stem were spalled.Through the polysaccharide staining of the slices,it was found that the starch granules in the“kranz anatomy”vascular bundle sheath cells of the healthy sugarcane plant leaves are large and dense,and are compactly arranged on the side close to the mesophyll cells,however,the starch in vascular bundle sheath cells of RSD was not granular but clustered,and showed a dispersion state,indicating that the photosynthetic carbon fixation was affected.?2?In situ PCR results showed that the colonization niches of pathogen Lxx in sugar cane were mainly xylem,phloem and procambia of stem,vascular bundle sheath cells,mesophyll cells of the“kranz anatomy”and xylem vessels in parallel veins,xylem,bundle sheath cells and peripheral mesophyll cell in main vein.?3?Lxx were successfully isolated and cultured,and on the 180th day after the inoculation for healthy sugarcane,the number of Lxx cells in the leaves of the RSD-infected group and the control group began to show a significant difference in frequency distribution?P<0.01?.On the 210th day after the inoculation,the median of Lxx population of RSD-infected group increased 242.83%compared that on the180th day,which indicating that Lxx was rapidly enriched in sugarcane leaves from the 180th to the 210th day.?4?The DEGs in the leaves of the RSD-infected and control groups were enriched in the photosynthetic carbon fixation pathway and chloroplast-related functions after inoculation.The expression levels of key enzyme genes in the C4pathway were all down-regulated,with the coding genes for PEPC,NADP-ME,and PPDK down-regulated by approximately 10,5,and 5 times respectively.?5?Compared with the control group,the activity of PEPC in the RSD-infected group was respectively decreased by 36.39%,52.89%,and 11.29%on the 150th,180th,and 210th day after inoculation.On the 180th and 210th day,the activity of PPDK respectively decreased by 25.00%and 36.12%,and the activity of NADP-ME was respectively decreased by 23.26%and 38.42%.The expression levels of PEPC and PPDK in the RSD-infected were lower that in the control group from the 150th day to the 210th day,the expression level of NADP-ME in the RSD-infected was significantly lower than that in the control group from the 120th to the 210th day.Summary:The infection of Lxx disrupts the integrity of vascular bundle sheath cells in the“kranz anatomy”of sugarcane leaves,resulting in irregular distribution of starch in vascular bundle sheath cells of leaves and affecting photosynthetic carbon fixation.Lxx-infection affects the expression of photosynthetic carbon fixation pathway genes in sugarcane,especially with the increase of Lxx cell numbers in leaves,the activities of the key enzymes PEPC,PPDK and NADP-ME and the expression abundance of the corresponding genes in the C4 cycle of sugarcane decreased,which affecting the ability of the C4 cycle to operate on CO₂,resulting in the reduction of photosynthetic carbon fixation capacity in sugarcane.