Figure 1.
Bioinformatic pipeline used for the identification of Hyaloperonospora arabidopsidis (Hpa) HaRxLs.
(*) The genome browser is maintained at the Sainsbury Laboratory (gbrowse2.tsl.ac.uk/cgi-bin/gb2/gbrowse/hpa_emoy2_publication).
Figure 2.
Hpa effector candidates (HaRxLs) were delivered on 12 Arabidopsis accessions through the bacterial TTSS of the Pst-LUX strain. Levels of bacterial growth were measured quantifying bioluminescence (photon counts) emitted by the bacteria present on whole plants. The ratio of the average photon counts per second (CPS) per gram of fresh weight (FW) emitted by the bacteria delivering a given HaRxL versus the bacteria delivering control proteins was determined per accession. Experiments were repeated at least three times and statistical tests applied. Results and conclusions are shown in Table S2 and Figure 3.
Figure 3.
Hpa HaRxLs can promote or decrease Pst-LUX growth in different Arabidopsis accessions.
The graph illustrates the outcome of the interaction between 12 Arabidopsis accessions (X axis) and Pst-LUX clones delivering 64 different Hpa effector candidates (HaRxLs, Y axis). Bars indicate the number of host accessions where the delivery of a given Hpa RxLR-like candidate effector by Pst-LUX conferred either enhanced (green bars), decreased (magenta bars) or no change (black bars) in bacterial growth, measured as bioluminescence, compared to the controls. The arrow indicates the threshold set up to consider that a given HaRxL truly enhances Pst-LUX bioluminescence. The asterisks indicate HaRxLs that suppress callose deposition in Col-0 when delivered via Pst-ΔCEL. High suppression levels are marked with (+). For details see Table S2, columns R,S and T. NC 1,2,3,4: negative internal controls.
Figure 4.
Suppression of PTI as a virulence tool for Hpa.
(A) Pre-treatment of Col-0 leaves with flg22 or Chitin reduces Hpa isolate Noco2 hyphal colonization. Leaves of four-week-old Col-0 plants were pre-infiltrated with 100 nM flg22, inactive flg22 (from A. tumefasciens) or Chitin (200 µg/ml) 24 h before inoculation of Hpa Noco 2 (5×104 sp/ml). Pictures show trypan blue stained leaves at 5 days post-Hpa spraying (dps). This experiment was repeated three times with similar results and also for Emoy2 on Oy-0 plants. Panels i, ii and iii: the whole area shown was pre infiltrated. Panels iv, v and vi: only the right side of the picture was infiltrated. Dotted vertical line indicates approximated infiltration boundaries. Bar is 500 µm. (B) Pre-Induced PTI responses reduce Hpa asexual reproduction. Leaves of three-week old Col-0 plants were infiltrated with the indicated solutions 24 h before infection with Noco2 (5×104 sp/ml). Conidiophores per leaf were counted on trypan blue stained leaves excised at 5 dps. Bars represent the average ±2× SE of 40 leaves. This experiment was repeated three times with similar results. (b) p value <0.01, T-test. (C) Hpa infected tissues show reduced ROS response to flg22. Leaf discs from uninfected and infected Col-0 plants were treated with 100 nM flg22, and the level of ROS generated measured with a Luminometer. Values indicated are average of Relative Luminescence Units (RLUs) ± SE of 24 leaf discs. (D) HaRxLs delivered by Pst-ΔCEL in Col-0 plants suppress callose deposition. Effector's impact on the level of Pst-ΔCEL-triggered callose deposition is presented in the Y-axis. The average reduction (in percentage) of callose deposits observed when a given candidate effector was delivered, compared to the number of callose deposits observed when control proteins were delivered by Pst-ΔCEL, is represented by the shapes in the body of the graph. HaRxLs were also categorized according to their phenotype on Pst-LUX bioluminescence in Col-0 (X-axis). The arrow indicates the threshold set up to consider callose deposition as significantly suppressed. The numbers in the body of the graph correspond to the percentage of HaRxLs able to suppress callose deposition among each bioluminescence category. (*) Indicates p<0.05 of Z-test versus random distribution expected for the number (n) of HaRxLs on each group.
Figure 5.
Arabidopsis Col-0 plants expressing constitutively HaRxLs support enhanced growth of P. syringae ΔavrPto/ΔavrPtoB and Hpa isolate Noco2.
(A) Four leaves of three five-week-old plants of two independent transgenic lines per HaRxL were infiltrated with Pst-ΔavrPto/ΔavrPtoB at OD600 = 0.0005. Bacterial growth was determined at 3 dpi by traditional growth curve assays. Bacterial populations immediately after inoculation (3 h; 0 dpi) were averaged among plants and are represented by the solid black horizontal line, with 2× SE represented by the dashed horizontal lines. (a) T-test p value<0.05, (b) T-test p value<0.01.This experiment was repeated two times with similar results. (B) Two-week-old seedlings were spray inoculated with a suspension of 1×104 conidiospores per ml of Hpa isolate Noco2. At 6 dps, whole seedlings were cut and stained with Trypan blue. The number of conidiophores per leaf was counted in 4 leaves per seedling. Ten seedlings were analyzed per transgenic line per HaRxL. The horizontal black and dashed lines represent the average ±2× SE of the number of conidiophores per leaf found in the hyper-susceptible mutant Col-0 eds1-2. (a) T-test p value<0.01, (b) T-test p value<0.05. This experiment was repeated three times with similar results.
Figure 6.
Arabidopsis plants expressing constitutively HaRxLs accumulate less ROS and/or callose in response to flg22.
(A) Leaf discs from four-week-old transgenic plants expressing the indicated HaRxL were sampled and floated in water 14 to 16 h prior to flg22 treatment. Photon emission was measured every 100 milliseconds for 40 minutes. Lines and error bars represent the mean of maximum values of photon counts ±2× SE of 24 independent leaf discs. This experiment was repeated four times with similar results. (B) Leaves of four-week-old transgenic lines were hand inoculated with 100 nM of flg22. Twenty-four hours post-inoculation, leaf discs were sampled and stained with aniline blue for visualization of callose dots. The bars represent mean ±2× SE of callose dots per image photographed (field of 0.22 square centimeters). Callose dots were quantified with ImageJ. Twenty leaf discs were analyzed per transgenic line. This experiment was repeated three times with similar results.
Figure 7.
Summary of phenotypes observed upon expression of HaRxL effectors in Arabidopsis.
Graphical comparison of the results obtained using the transient EDV assays and stable constitutive expression for nine different HaRxLs. The phenotypes analyzed include bioluminescence of Pst-LUX, suppression of callose deposition triggered by Pst-ΔCEL, growth of Pst-ΔavrPto/ΔavrPtoB, suppression of ion leakage triggered by delivering AvrRPM1 via Pf0-1, growth (conidiation) of Hpa compatible (Noco2) and incompatible (Emoy2) isolates, and suppression of the levels of ROS and callose deposition triggered by flg22 treatments.