SRK And FERONIA Receptor Kinases Control Distant Hybridization Reproductive Barriers In The Brassicaceae

Distant hybridization is an effective way to innovate germplasm resources and develop new varieties,but is hindered by interspecies reproductive barriers.Therefore,it is essential to reveal the molecular mechanisms regulating the reproductive isolation of distant hybrids and develop effective means to break down interspecies breeding barriers.In the Brassicaceae,interspecies pollen is unilaterally inhibited on SI stigmas but accepted on stigmas of self-compatibility（SC）.In this research,we use heading Chinese cabbage,a subspecies of Brassica rapa,and Arabidopsis thaliana as SI and SC stigma,respectively,and pollinate them with pollen from Brassica rapa,Brassica oleraceae,Barbarea vulgaris and Arabidopsis thaliana.We used molecular biology,biochemistry,genetics and physiology to investigate the mechanisms of reproductive isolation in distant hybrids.The main findings are as follows:Stigma ROS is an important factor in the formation of distant hybridization barriers.Aniline blue staining revealed the similarities between distant incompatibility and self-incompatibility,i.e.,difficulty of pollen tube germination on the stigma as well as growth.By examining the ROS production in B.rapa stigmas after pollination with different pollen,it was found that pollen from intraspecies as well as interspecies all could cause stigmatic ROS increased.By reducing ROS with the ROS scavenger,self-incompatibility in B.rapa mature stigma was effectively break down and as well as distant hybridization of B.rapa×B.ole and B.rapa×B.vul at B.rapa bud stage.These results suggest that stigmatic ROS is important for the formation of distant hybridization barriers.SRK controls rejection of UI pollen.By knock down the function of SRK in B.rapa using antisense oligonucleotides（AS-Br SRK46）or using a Br SRK transmembrane structural domain deletion mutant line,we found the SRK deficiency allowed B.ole pollen tubes to pass through the mature stigmas of B.rapa and B.vul pollen tubes to enter the bud stage stigmas of B.rapa.ROS detection showed that Br SRK-deficient suppressed the increase of ROS caused by self-incompatible pollen,B.ole pollen which pollinated at mature stigmas and B.vul pollen at bud stage.By pollinated the Arabidopsis thaliana,Brassica rapa,Brassica ole and Barbarea vulgaris pollen onto the stigma of self-incompatible Arabidopsis thaliana-S₁₃/WT（SI）,we also observed the ROS increase and pollen growth arrest,further confirming that SRK controls rejection of UI pollen by regulating stigmatic ROS.UI and SI activate SRK-FER-regulated ROS.AS-Br FER1 treatment,or SI Arabidopsis thaliana(S₁₃/WT)was crossed into fer-4 background silenced Br FER1 expression in Brassica rapa stigmas,and the results of aniline blue revealed that intraspecific and interspecific pollen was growth into Brassica rapa or S₁₃/fer-4 stigmas;by ROS staining,it was found that AS-Br FER1 treatment or S₁₃/fer-4 inhibited ROS elevation caused by intraspecific and interspecific pollen.It is suggested that FER controls rejection of UI pollen by regulating the stigma ROS.We further found SRK interact with FER directly by Y2H,pull-down and Bi FC.In addition,adding SI and UI pollen extracts or SCR46 enhance the interaction and increase ROS.The results indicated that the SCR46 increase the ROS accumulation by activating the FER-RBOHs signalling pathway.FER and PCP-Bs favour SC over UC pollen.In fer and rbohd were used for half self-pollination and half distant pollen pollination,the mutants had a reduced blocking strength to the distant pollen.The absence of FER and RBOHD was shown to promote interspecific pollen hydration and pollen tube growth.In the treatment of A.thaliana stigmas with At PCP-Bγreduced ROS at a faster rate than treatment with Br PCP-B3.It was demonstrated by pull-down that both At PCP-Bγand Br PCP-B3 interacted with of At FER（ED）,and At PCP-Bγcompetes for the interaction of Br PCP-B3 with At FER（ED）.In contrast,Br PCP-B3 did not affect the interaction of At PCP-Bγwith At FER（ED）.It suggests that PCP-Bs of the same species bind more effectively to FER.Compatible pollen induced-NO reduces ROS.It was found that the average fluorescence intensity of NO peaked at 15 MAP in Brassica rapa and at 5 MAP in Arabidopsis thaliana.In the treatment of A.thaliana stigmas with At PCP-Bγincrease NO at a faster rate than treatment with Br PCP-B3.Contrary to the changes in ROS.It reveals an inverse relationship between NO and ROS in distinguishing between interspecific compatible pollen.In fer-4,NO did not respond to elevated after compatible pollination compared to WT.The results suggest that the elevation of NO is dependent on FERONIA.In addition,NO donor GSNO resulted in a significant decrease in ROS and allowed UI pollen growth into Brassica rapa stigma.The NO scavenger c PTIO treatment showed the opposite result.In genetics,ROS was higher in noa1 and pollen hydration and pollen tube growth was slower compared to the wild type（WT）,while in hot5-4 showed lower ROS levels and significantly faster pollen hydration and pollen tube growth compared to the wild type（WS）.These suggests that NO affects pollen hydration and pollen tube growth by ROS.NO nitrosates FER in compatible response.Br FER1（KD）is nitrosylated in vitro by treatment with the NO donor GSNO and decreases the interaction of Br FER1 and Br ROP2;Br FER1（KD）Cys730 is a key site for nitrosylation as detected by protein profiling,and Br FER1^C730W（KD）reduces the interaction of Br FER1 with Br ROP2.The Arabidopsis overexpression line,p At FER::At FER-GFP,reduced the interactions between At FER and At ROP2 in vivo after compatible pollination;Br RBOHs were similarly nitrosylated.Breaking the barrier for distant breeding.The hybrid embryos were successfully obtained by overcoming distant hybrid incompatibility through chemical treatment with ROS,NO,SRK and FER as targets,such as the ROS scavenger Na-SA and the NO donor GSNO,or by AS-Br FER1,AS-Br SRK46 and AS-Br RBOHF to block the regulatory pathway of ROS.This study provides an in depth and systematic analysis of the regulatory mechanisms of self-incompatibility and distant hybrid incompatibility in Brassicaceae,and develops effective means to break down interspecies breeding barriers,as well as theoretical guidance and technical support for germplasm resource innovation and new variety breeding.