The Role Of Electrical And ROS Signals In Jasmonates-Regulated Resistance Against Meloidogyne Incognita In Tomato

In recent years,with the rapid expansion of the cultivated area of facility vegetables in China and the gradual increase of continuous cropping years,root-knot nematodes have become a major obstacle to the current production of facility vegetables,causing huge losses to the agricultural economy.The loss caused by plant parasitic nematodes can reach 100 billion dollars annually,of which the loss caused by root-knot nematodes is as high as 70%.Studying the resistance mechanism of root-knot nematode represented by tomatoes and solving the bottlenecks restricting the production of facilities in China are problems that need to be solved urgently.Based on this,analyzing the response mechanism of tomato to root-knot nematodes by molecular,physiological,biochemical and environmental regulation methods to improve tomato resistance,increase tomato yield,reduce use of pesticides,increase economic benefits,protect the cultivation environment and guarantee food safety is of great scientific and practical significance.The vegetable crop tomato is used as the research material by using genetics,physiology,molecular biology,biochemistry and other means in this thesis.We clarified that RKN induces a systemic transmission of electrical and ROS signals.We found that GLRs-dependent electrical activity and RBOH1-dependent ROS production are critical for JAs synthesis in leaves and RKN resistance.We reported that crosstalk between cytoplasmic electrical activity and ROS production is intrinsic to long-distance signal transmission.We showed that activation of MPK1/2 is involved in the induction of JAs synthesis.Major results are shown below:1)RKN induces a systemic transmission of electrical and ROS signals.RKN induced an increase in the accumulation of JAs in both the leaves and roots,particularly at 24 h hpi.A 48 h continous recording revealed that RKN induced intermittent changes in the surface potential of stems,petioles and leaf lamina and the cytoplasmic potential in the leaf cells of all plants with intervals of minutes to hours.Histochemical analysis with DAB staining revealed that RKN infection induced an accumulation of H₂O₂ in the vascular systems of roots,stems and petioles.Quantitation of DAB staining intensity showed RKN-induced H₂O₂ accumulation was highest at 24 hpi and decreased from the roots to the petioles.RKN infection induces the generation and systematic transmission of electrical and ROS signals in tomato.2)GLRs-dependent electrical activity is critical for leaf JAs synthesis and related defenses.The pTRV-GLR3.3 and pTRV-GLR3.5 plants both showed significantly lower resistance to RKN,together with decreased JA accumulation in the leaves relative to pTRV plants.The grafted plants with glr3.5 as rootstock or scion showed decreased electrical activity,together with reduced resistance against RKN relative to self-grafted WT plants and RKN-induced accumulation of JAs in the leaves and roots was attenuated.When a segment of the glr3.5 stem was inserted into the WT stem?WT/glr3.5/WT?,there was a significant decrease in the resistance to RKN.It attenuated RKN-induced changes in electrical pulse amplitude and duration and decreased the accumulation of JAs in the leaves and roots relative to self-grafted WT plants?WT/WT/WT?.Other experiments showed that artificial current injection on the stem surface significantly decreased the number of RKN galls and increased the accumultation of JAs in the plants.The GLR3.5-dependent electrical signals mediate JAs synthesis to regulate RKN resistance.3)RBOH1-dependent ROS production is important in the regulation of leaf JAs synthesis and RKN resistance.RKN-induced accumulation of H₂O₂ was associated with an increase in the activity of NADPH oxidase in the leaves.qRT-PCR analysis revealed that RBOH1 was the most highly expressed than other 7 RBOHs.Plants with rboh1 as scion or rootstock showed decreased resistance to nematode infestation.No substantial increases in H₂O₂ accumulation were observed in the stems of rboh1/rboh1plants and accumulation of H₂O₂ only in the rootstock stems but not scion stems of the rboh1/WT plants.H₂O₂ accumulation in the apoplast and/or in the leaf tissues and accumulation of JAs in the leaves or roots were abolished in plants with rboh1 as the rootstock or scion.The grafted plants with a segment of rboh1 stem significantly reduced resistance to RKN infestation and compromised RKN-induced accumulations of H₂O₂ in the stem above the rboh1 graft and JAs accumulation was not observed in the leaves or roots.The foliar application of H₂O₂ induced JA accumulation in the leaves.The RBOH1-dependent ROS signals mediate JAs synthesis to regulate RKN resistance.4)Crosstalk between cytoplasmic electrical activity and ROS production is intrinsic to long-distance signal transmission.We found RKN-induced increases in NADPH oxidase activity in the leaves were compromised in plants co-silenced for GLR3.3 and GLR3.5.RKN-induced accumulation of H₂O₂ in the leaf tissues and in the apoplast of the leaves or in the stems was attenuated in the grafted plants with glr3.5 as rootstock or scion.RKN infestation induced H₂O₂ accumulation in the WT rootstock stems but not in the glr3.5 stem segments or the WT scion stems of the WT/glr3.5/WT plants.H₂O₂ accumulation was not induced in the apoplast of the leaves or in the whole leaves in response to RKN infestation in the shoots of the WT/glr3.5/WT plants.The grafted plants with rboh1 as rootstock or scion,or those with an inserted rboh1 segment showed attenuated RKN-induced electrical activity with decreased pulse amplitude and duration.We applied current injection to the stems which induced resistance and accumulation of H₂O₂ in the the vascular system of the shoots and the aploplast of the leaves of the WT plants,but this was not observed in the glr3.5 or rboh1 plants.The interaction between electrical and ROS signalings regulate RKN resistance together.5)Activation of MPK1/2 is involved in the induction of JAs synthesis.RKNs induced MPK1/2 activation from 3-6 hpi and MPK1/2 activation reached a peak at 24 hpi.While RKN infection induced the activation of MPK1/2 in the leaves of WT/WT/WT plants,this activation was,however,attenuated in the leaves of the WT/rboh1/WT plants and the WT/glr3.5/WT plants.Current injection-induced activation of MPK1/2 was significantly attenuated in the leaves of the rboh1 and glr3.5 mutants.The susceptibility to RKN infestation increased in MPK1 and/or MPK2-silenced plants and RKN-induced accumulation of JAs was compromised in the leaves and roots.MPK1/2 regulates JAs synthesis and RKN resistance in a manner independent of JA synthesis gene expression.It is concluded that the RKN induced the systemic transmission of electrical and ROSsignals from attacked tomato roots to the leaves leading to an increased accumulation of JAs in the leaves.GLRs-dependent electrical activity and RBOH1-dependent ROS signal are critical for leaf JAs synthesis and related defenses.Moreover,activation of MPK1/2 is involved in the induction of JAs synthesis.These findings reveal a systemic signaling loop that integrates electrical,ROS and JAs signals to enhance the resistance in distal organs via root-shoot-root communication.