This paper answers the question: how to activate the downstream immune response at the single cell level when the root system of Arabidopsis thaliana is facing damage and pathogen infection.
Identifying the molecular patterns associated with pathogens is very important for plant immune response. This complex sensing system is constantly exposed to microbial communities, and how to effectively apply it to roots is still a mystery. By observing the expression pattern and reaction level of MAMP receptor in Arabidopsis roots at the cellular level, we observed that the outer layer of differentiated cells showed a low level of PRR (pattern recognition receptor) expression and lacked the downstream MAMP reaction. However, these cells can respond through the damage of neighboring cells. Laser ablation of small cell clusters can strongly up-regulate the PRR expression of neighboring cells, and the increased PRR level is enough to cause immune response of unresponsive cells. Finally, local injury can also lead to immune response to non-immunogenic beneficial bacteria. Damage gating is covered by receptor overexpression, which makes the pathogen unable to colonize in the rhizosphere. Our study found that cell injury can "turn on" local immune response, which is helpful to understand how cell injury occurs. Although there are microbial patterns in the soil, MAMP can feel them.
The background is briefly introduced. Such as the labeling process of PTI, such as the accumulation of ROS, the activation of MAPK and the expression of other marker genes.
MATERIALS: Triple M Venus fused to nuclear localization signal (NLS-3xm Venus), a fluorescent labeling material, which can analyze the reaction of MAMP in vivo and at the cellular level.
Mammp reaction marker gene: 1. PER5 (peroxidase 5), induced by early MAMP, is highly expressed in roots; 2.WRKY 1 1 (WRKY DNA binding protein11); 3.MYB5 1(MYB domain protein 51); 4. frk 1 (receptor-like kinase induced by FLG 221).
MAMP response induced by flg22 in Arabidopsis roots is limited in space.
From the ABC of Figure 1, it can be seen that among the four marker genes, PER5 and FRK 1 treated with flg22 have lower background noise and better induction, and the location of MAMP reaction is limited to meristem and elongation region. For the differentiated root part, the response signal of MAMP could not be observed even with high concentration of flg22 treatment. (Red is PI staining, and green is fluorescence signal)
In order to accurately evaluate the signals of specific cells and cell types, the author prepared the genetic material of two markers, and the other was RFP which was constitutively expressed and targeted to plasma membrane. In the long-term observation, the author found that the differentiated roots showed weakened and limited space MAMP reaction after flag22 treatment, and during the formation of lateral roots, the cells near the lateral root primordium were destroyed due to the ejection of the lateral root primordium. At this time, when flg22 was applied, the cells near the lateral root primordium showed a strong reaction to MAMP treatment (1D, 1F, and the blue arrow indicates the lateral root primordium. The dotted circle indicates the formation of lateral root primordium, and the white arrow indicates MAMP reaction. Moreover, when spontaneous and non-induced cell death (1E*) occurred, the MAMP response induced by flg22 was found in neighboring cells, which was related to the receptor (FLS2) (1G). Therefore, differentiated roots have the ability to respond to MAMPs, which is induced by highly localized ways (lateral root primordium formation, spontaneous cell death).
Laser-induced cell ablation causes local MAMP response in roots
(F2A white asterisks represent laser ablated cells, all of which play a role in differentiated roots, EP epidermis, Co cortex, En endodermis and St stele cells.)
After laser ablation destroyed different types of root cells, we only observed a strong increase in flg22 reaction in neighboring cells. However, for the ablated cells themselves, MAMP marker genes were not or rarely induced, which revealed that the destruction of their own cells was not enough to cause a strong MAMP response. After the epidermis, cortex and endodermis were ablated, stele cells reacted strongly to flag22, but the ablation of epidermal cells did not cause the cells near epidermis to react to flag22 (F2AB).
Earlier, we knew that beneficial microorganisms in the soil would colonize the elongation area of the heel. This process was simulated by treating the extension zone (F 1B) with high dose (1um) of flg22, which could cause MAMP reaction without damage. In order to rule out the dose effect, we want to know whether low dose (100 nm) of FLG 22 combined with cell ablation will cause MAMP reaction in neighboring cells. We found that only a small amount of MAMP-induced reaction (F2CD) was observed in the undamaged elongation zone after the application of flag22 of 100nm, but if the epidermal cells in the elongation zone were destroyed, the MAMP reaction of cortical cells was enhanced, which was similar to that of differentiated roots (F2ABCD). Therefore, the authors speculate that the enhancement of MAMP response in neighboring cells caused by injury may occur in the whole root.
DAMPs alone is not enough to induce MAMP reaction
At this point, the author thinks, is it possible that the damping caused by cell damage leads to the MAMP reaction of neighboring cells? Therefore, the author selected several classic DAMPs, such as atpep 1, ATP, cellobiose, OGS, and the mixture of the above four DAMPs for cocktail processing. After mixing each DAMPs with flg22, it was found that even using high concentrations of DAMPs could not induce a strong and sustained reaction to flag22. [EZ is fluorescent because high concentration (1um) of Flag22 will cause MAMP reaction, and no cell damage is needed]. ATPEP 1 is an exception, which can only cause weak FRK 1 reaction in DZ region, but not PER5 reaction. This shows that the perception of injury signals by neighboring cells is more complicated than the author imagined, not through DAMPs, but through the release of ions and permeates or other mechanical pressures.
The expression of MAMP receptor is induced by cell ablation and is sufficient to induce reactivity.
The author found that the FLG202 reaction (F4A) could be observed in the differentiated outer root cell layer by applying FLG202 treatment in FLS2 overexpression materials, which suggested whether the MAMP reaction of neighboring cells caused by cell damage was related to the increase of PRR expression.
We found that when roots were damaged, the transcription level of FLS2 was activated in both mature and elongation regions (F4, BDFG). Moreover, the up-regulation degree of FLS2 in time and space is consistent with the reaction pattern observed by MAMP(FRK 1 and PER5) (F2ABCD is compared with F4BCDF). Starting from F4E, after receiving cell injury and applying flag22, we found that the local expression level of FLS2 increased. The author wanted to know whether the activation of FLS2 was related to MAMP reaction, and found that when treated with the same flag22 and cell injury (F4HI), the cells adjacent to MAMP reaction were consistent with those activated by FLS2. This also shows that cell injury induces high expression of PRR, which is enough to cause MAMP reaction.
Flg22 reaction in differentiated roots by kjeldahl band division
(sgn3-3 Kjeldahl band deletion mutant, foreign substances can directly enter the stele sheath cells. F5AB are all differentiation regions)
FLS2 is highly expressed in stele sheath cells, but in the mature region of sgn3-3, when FLG2 is applied, no response of FLG2 is observed. However, in the over-expressed FLS2 material, the FLG2 reaction was observed in sgn3-3, but not in the wild type (F5AB, the red arrow indicates the stele sheath cells, and PI could not stain them due to the obstruction of Kjeldahl band. This result shows that Kjeldahl band can be divided into immune perception. In WT, the amount of FLS2 can not cause the reaction of MAMP, but the strong promoter can (F5. AB column 3).
Cork lamellae interfere with the perception of flg22 in the inner cortex
Sgn3: Kjeldahl band is discontinuous at the 25-cell stage after the start of extension.
Esb 1: The Kjeldahl zone was discontinuous at the 25th cell stage after the extension began, and endoderm was embolized in advance.
ABA treatment: endoderm embolism in advance.
The function of Kjeldahl zone is to prevent foreign substances from entering the stele cells, and the inner plug layer (a secondary cell wall modification) finally surrounds the whole endoderm, which inhibits the molecular absorption of the endoderm, because the hydrophobic inner plug layer does not allow molecules from the cell wall to enter the cell membrane (F5, CD) of the endoderm.
We found that early differentiated endothelial cells (25 cells after initial extension without embolism) showed a response to flg22 in FLS2 overexpression materials, but late differentiated endothelial cells (55 cells after initial extension with embolism) did not show a response to flg22. (F.5E, column 2) In cells treated with esb 1 and ABA, the response of flg22 was inhibited in the early endoderm, which indicated that the cells were protected by premature embolization and FLS2 could not bind to flg22 (F.5E). This phenomenon occurs even when the epidermis and cortex are destroyed in the root mature area (FLS2 is separated from flg22 due to premature endoderm embolism) (F.5F).
Cell injury activates the expression of multiple pattern recognition receptors.
After completing the mechanism of a receptor, the author wants to expand the mechanism, and then finds three kinds: PRR, EFR, CERK 1 and RLP23. The authors observed that all three receptors were up-regulated under the condition of cell injury, which indicated that cell injury mediated a very common up-regulation reaction to MAMPs. (F6AB)
After sorting out the classical LRR domain receptors, the author wants to know whether it is also suitable for non-classical receptors, such as LORE (lipooligosaccharide specificity reduction elicitor). Similarly, after the early differentiated cells were destroyed, the expression of LORE was strongly induced, and the application of its ligand also dominated the MAMP reaction in the extension region, but not in the mature region, which was similar to flg22 (F6D). This extends this mechanism to other PRRs.
Local control of immune response by damage in root-bacteria interaction
Just now, it was a cytological test, which will eventually be put into practice. Using beneficial bacteria (CHA0) to infect roots, the author observed that although there was strong colonization in roots, no MAMP reaction was observed in undamaged differentiated roots. Similar to cytological experiments, MAMP reaction (spontaneous destruction) was observed in adjacent cells formed by lateral root primordia, and FLS2 overexpression could cause MAMP reaction without damaging cells (F7A). When bacterial infection is combined with cell ablation, cells near the destruction site show a reaction to MAMP in the presence of bacteria (F7BC). Bacteria provide a variety of ligands.
Then the author used bacteria harmful to rhizosphere, GMI 1000 and GMI 1000. Initial infection will not cause cell destruction and strong MAMP reaction, but persistent infection will lead to the death of some epidermal cells. At this time, an increase in local MAMP reaction can be observed in neighboring cells (F7D). In the mature region of FLS2 overexpression material, both flg22 treatment (F4A) and beneficial bacterial infection (F7A) showed MAMP reaction and low bacterial growth (F7A, e). Interestingly, the flagellin of harmful bacteria GMI 1000 could not activate the FLS2 receptor in Arabidopsis thaliana.
See F7F for the pattern diagram proposed by the author, so I won't repeat it.
The inspiration for me
1. Make the experiment detailed, the big framework has been built, and the details depend on success or failure.
2. do more approximate equations, such as A=B, B=C, then a = C.
Every step of the experiment needs to be done, and you can't be lazy and take it for granted. After all, organisms are so complicated ~