Muons penetrate much further than X-rays, they do essentially zero damage, and they are provided for free by the cosmos.
Image credit: Science Source/Digital Globe.
Contributed by Brian J. Staskawicz, December 13, 2016 (sent for review November 23, 2016; reviewed by Sheng Yang He and Bing Yang)
Article Figures & SI
Figures
- Fig. S1.
XgΔavrHah1 has an in-frame deletion of the DBD. (A) Schematic of the in-frame deletion that removes the DBD from avrHah1 to create avrHah1ΔDBD. NLS, nuclear localization signal. (B) Southern blot analysis of BamHI-digested genomic DNA from X. gardneri strains listed probed for the DBD (Top) or the T3SS (Bottom). The size of BamHI-digested avrHah1 is 2,964 bp, and the size of BamHI-digested DBD deletion of avrHah1 is 1,491 bp. (C) Cell death (or HR) occurs in response to activation of the Bs3 resistance gene in pepper 30R and appears here as a dark, opaque area. XgΔavrHah1 does not activate Bs3 and is pathogenic on pepper 30R. Complementation of XgΔavrHah1 with avrHah1 or avrBs3 restores activation of Bs3 resistance and HR in pepper 30R.
- Fig. 1.
AvrHah1 promotes the intake of water into the apoplast of X. gardneri (Xg)-infected plants. N. benthamiana was syringe-infiltrated with X. gardneri (1), XgΔhrcV (2), XgΔavrHah1 + avrHah1 (3), and XgΔavrHah1 (4) at OD600 = 0.1 [depicted in the diagram as the bottom left area of the leaf, top left area of the leaf (2), bottom right area of the leaf (3), and top right area of the leaf (4), respectively]. At 48 hpi, a 30-μL drop of water was pipetted on top of a small epidermal wound in the infiltrated areas. Pictures were taken at the times indicated in each frame until 2 min and 20 s after application of the first water drop.
- Fig. 2.
AvrHah1 induces water soaking in tomato, whereas AvrBs3 activates a hypersensitive response. Tomato Heinz 1706 leaves were syringe-infiltrated with the X. gardneri (Xg) strains indicated (OD600 = 0.1). At 48 hpi, leaves were submerged in water for 20 min, blotted with a Kimwipe, and photographed.
- Fig. 3.
AvrHah1 enhances water soaked lesions in Xe85-10. Tomato leaves were infiltrated at 104 CFU/mL with Xe85-10 alone (−) or Xe85-10 complemented as indicated and observed at 6 dpi. Plants were grown at ambient humidity and were not submerged in water before observation.
- Fig. S2.
XgΔavrHah1 does not experience an in planta growth defect in tomato. Tomato leaves were infiltrated with X. gardneri strains at 104 CFU/mL. At 6 dpi, six leaf discs per strain of X. gardneri were sampled for in planta bacterial growth.
- Fig. 4.
Water soaking can introduce bacterial cells into the apoplast. N. benthamiana was syringe-infiltrated with either X. gardneri (Xg) or XgΔhrcV at OD600 = 0.1. Water soaking was induced at 48 hpi with a 30-μL drop of Tet-resistant X. gardneri (X. gardneri TetR, 105 CFU/mL in 1 0 mM MgCl2) on a wound (arrow). Leaf discs were collected away from the wound site after 5 min (dashed circle), ground in 10 mM MgCl2, and dilutions were plated on either Rif or Rif + Tet to select for the growth of all X. gardneri or the X. gardneri TetR from the water-soaking inoculum, respectively.
- Fig. 5.
AvrHah1 and AvrBs3 are differentially recognized by tomato Bs4. Tomato leaves were infiltrated with the Xe85-10 strains indicated at OD600 = 0.1 and observed 48 hpi. Cell death is visible in response to delivery of avrBs3, whereas the beginnings of water soaking are apparent in response to avrHah1. Leaves were left at ambient humidity and were not submerged in water before observation.
- Fig. S3.
AvrHah1 has unique and overlapping targets with AvrBs3. AvrHah1 (Top) can activate expression from the Bs3 promoter (proBs3) using an overlapping EBE with that of AvrBs3 (Bottom). The predicted AvrHah1 EBE is in purple, whereas the AvrBs3 EBE has two nucleotide extensions on either side marked in yellow. Similar RVDs between AvrHah1 and AvrBs3 are depicted by green highlighting.
- Fig. 6.
Tomato gene activation in response to AvrHah1. (A) Semiquantitative RT-PCR in tomato inoculated with the indicated X. gardneri (Xg) strains (24 hpi; OD600 = 0.25); * indicates genes with predicted promoter EBEs for AvrHah1 (e.g., proposed direct targets). (B) Transient luciferase reporter assay in N. benthamiana. Promoter::luciferase constructs are displayed along the x axis. Expression binary vectors carrying AvrHah1 or the bHLH transcription factors are listed on the left, and an “X” signifies codelivery with a promoter::luciferase reporter; * indicates significantly different luciferase activity from promoter background (codelivered with empty Agrobacterium) (P < 0.001).
- Fig. 7.
dTALEs demonstrate that the bHLH transcription factors and the PL are S genes of AvrHah1. (A) Semiquantitative RT-PCR in tomato infected with XgΔavrHah1 complemented with dTALEs for the bHLH transcription factors (dT bHLH3 and dT bHLH6), the PL (dT PL), and the PE (dT PE) (24 hpi; OD600 = 0.25). (B) Quantitative water-soaking measurements were obtained by centrifugation and weighing of apoplastic fluid from infected tomato leaves (48 hpi; OD600 = 0.1; 20 min water bath). Average weights and SEs from 12 samples (each consisting of two 0.5-cm2 leaf discs) are shown.
Tables
- Table S1.
Information on dTALE construction to activate expression of bHLH3, bHLH6, PL, and PE
dTALE ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Score bp from ATG dT bHLH3 A T A G A T A T A A G C T A C C A G 5.45 274 NI NG NI NN NI NG NI NG NI NI NN HD NG NI HD HD NI NN dT bHLH6 A T A C A G G A T A T C C C T T T C 4.96 166 NI NG NI HD NI NN NN NI NG NI NG HD HD HD NG NG NG HD dT PL A C T A A A A G T A T T C A C A T C 4.22 381 NI HD NG NI NI NI NI NN NG NI NG NG HD NI HD NI NG HD dT PE A T T T C C C T C A C T A A T A C T 3.73 420 NI NG NG NG HD HD HD NG HD NI HD NG NI NI NG NI HD NG Binding scores were calculated using TALE-NT (27).
- Table 1.
Differentially up-regulated genes from an RNA-seq experiment comparing X. gardneri- and XgΔavrHah1-infected tomato
Soly Gene ID EBE score/bp from ATG Mean RPKM Predicted protein function X. gardneri XgΔavrHah1 Solyc02g070210 15.01/84 36.11 0.06 Phosphatidylinositol transferase Solyc02g084010 99.00 0.14 Auxin-induced SAUR-like Solyc02g089350 997.81 0.78 Gibberellin regulated Solyc03g033590 55.85 0.14 Auxin-induced SAUR-like Solyc03g097820 3.96/108 1,267.09 0.75 bHLH Transcription Factor Solyc03g114430 133.86 0.27 Unknown Protein Solyc03g116060 155.13 0.14 Gibberellin-regulated Solyc04g017720 550.74 0.89 Gibberellin regulated Solyc04g079700 13.26/173 16.26 0.07 WD-40 repeat family Solyc04g079860 39.32 0.13 Glycosyltransferase family GT8 Solyc04g081870 442.81 1.38 Expansin Solyc05g014000 206.02 0.25 Pectate lyase Solyc06g067910 95.26 0.28 Unknown function DUF642 Solyc06g068360 99.04 0.4 Ethylene-resp. transcription factor 7 Solyc06g071930 461.79 2.81 Unknown Protein Solyc06g072520 9.21/139 568.68 0.38 bHLH Transcription Factor Solyc07g006310 129.78 0.24 Transcription factor Solyc08g062450 8.2/105 14.70 0.14 class II heat shock Solyc08g068720 13.74/220 196.13 0.01 Tyramine hydroxycinnamoyl transferase Solyc08g079780 13.96 0.08 Blue copper protein Solyc11g011210 1,436.40 8.86 Gibberellin regulated Solyc11g019910 89.27 0.22 Pectinesterase Solyc11g067180 94.81 0.15 Fatty acyl CoA reductase Solyc12g009840 156.10 0.64 Pyrophosphate-energized proton pump Mean reads per kilobase of transcript per million mapped reads (RPKM) of three biological replicates at 48 hpi are displayed. Genes are organized by Soly gene ID. Genes with predicted promoter EBEs for AvrHah1 are indicated with the score (best possible is 3.76) and distance from the start codon.
Data supplements
Supporting Information
- Download Supporting Information (PDF)
- Download Movie_S01 (MOV) - AvrHah1 promotes the intake of water into the apoplast of X. gardneri-infected plants. N. benthamiana was syringe-infiltrated (circled areas) with X. gardneri (1), XgΔhrcV (2), XgΔavrHah1 + avrHah1 (3), and XgΔavrHah1 (4) (OD600 = 0.1) (1, bottom left area of the leaf; 2, top left area of the leaf; 3, bottom right area of the leaf; 4, top right area of the leaf, respectively). At 48 hpi, a 30-μL drop of water was pipetted on top of a small epidermal wound in the infiltrated areas (time 0:00). Water soaking was allowed to progress for 2 min and 20 s. (The time progression is depicted in individual frames in Fig. 1.)
Water soaking by an indirect TAL effector target
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