The floral organ abscission pathway is necessary for pathogen-triggered leaf abscission.

<p>(A) Photos of cauline leaf AZ of WT plants and floral abscission defective mutants treated with DC3000 for 3 days. (B) Percent cauline leaves abscised 3 days after infection. Data are mean ± s.e.m.; n = 6 biological replicates (one plant each); letters indicated different statistical quantities t-test <i>P</i> < 0.05. (C) Micrograph of cauline leaf AZs from <i>hae hsl2</i> plants treated with or without DC3000. Images are representative from at least 4 replicates. Red arrows indicate enlarged AZ cells. Scale bar is 0.5 mm.</p

Leaf shedding as an anti-bacterial defense in Arabidopsis cauline leaves

<div><p>Plants utilize an innate immune system to protect themselves from disease. While many molecular components of plant innate immunity resemble the innate immunity of animals, plants also have evolved a number of truly unique defense mechanisms, particularly at the physiological level. Plant’s flexible developmental program allows them the unique ability to simply produce new organs as needed, affording them the ability to replace damaged organs. Here we develop a system to study pathogen-triggered leaf abscission in Arabidopsis. Cauline leaves infected with the bacterial pathogen <i>Pseudomonas syringae</i> abscise as part of the defense mechanism. <i>Pseudomonas syringae</i> lacking a functional type III secretion system fail to elicit an abscission response, suggesting that the abscission response is a novel form of immunity triggered by effectors. <i>HAESA</i>/<i>HAESA-like 2</i>, <i>INFLORESCENCE DEFICIENT IN ABSCISSION</i>, and <i>NEVERSHED</i> are all required for pathogen-triggered abscission to occur. Additionally <i>phytoalexin deficient 4</i>, <i>enhanced disease susceptibility 1</i>, <i>salicylic acid induction-deficient 2</i>, and <i>senescence-associated gene 101</i> plants with mutations in genes necessary for bacterial defense and salicylic acid signaling, and NahG transgenic plants with low levels of salicylic acid fail to abscise cauline leaves normally. Bacteria that physically contact abscission zones trigger a strong abscission response; however, long-distance signals are also sent from distal infected tissue to the abscission zone, alerting the abscission zone of looming danger. We propose a threshold model regulating cauline leaf defense where minor infections are handled by limiting bacterial growth, but when an infection is deemed out of control, cauline leaves are shed. Together with previous results, our findings suggest that salicylic acid may regulate both pathogen- and drought-triggered leaf abscission.</p></div

Bacteria with a viable type III secretion system can activate HAE expression and trigger cauline leaf abscission.

<p>(A) Plants expressing HAE-YFP (driven by the <i>HAE</i> promoter) were infected with virulent or avirulent DC3000. Images are 2 days after infiltration. The same samples are shown in the top and bottom panels where the top panels are imaged with reflected white light and the bottom panels are imaging YFP fluorescence. (B) Abscission of cauline leaves three days after infection. Data are mean ± s.e.m.; n = 5 biological replicates (one plant each); t-test versus MgCl₂ control; <i>*P</i> < 0.005. (C) Hypersensitive response in cauline leaves 20 h after infection. Data are mean ± s.e.m.; n = 7 biological replicates (one plant each, 2 leaves per plant). (D) Leaves infected with DC3000 or DC3000 that does not produce coronatine (COR^-) for two days. (E) Abscission of cauline leaves 3 days after infection. Data are mean ± s.e.m.; n = 8 biological replicates (one plant each). Scale bar is 0.5 mm.</p

<i>HAESA</i> is co-expressed with <i>PAD4</i> and <i>EDS1</i> in a number of different tissues and treatments.

<p>Publicly available microarray data indicates that <i>PAD4</i> and <i>EDS1</i> are statistically increased during the abscission process in stamen abscission zones [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007132#pgen.1007132.ref027" target="_blank">27</a>]. Furthermore, <i>HAE</i>, <i>PAD4</i>, and <i>EDS1</i> expression are up in shoot and leaf tissues of mutants with increased SA levels and down in mutants with decreased SA levels [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007132#pgen.1007132.ref055" target="_blank">55</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007132#pgen.1007132.ref056" target="_blank">56</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007132#pgen.1007132.ref031" target="_blank">31</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007132#pgen.1007132.ref057" target="_blank">57</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007132#pgen.1007132.ref058" target="_blank">58</a>].</p

Abscission occurs when DC3000 touches the AZ, however, long-distance signals are also sent from distal portions of the leaf to the AZ.

<p>(A) Leaves infiltrated in indicated portions of the leaf shown 3 days after infection. The same samples are shown in the top and bottom panels where the top panels are imaged with reflected white light and the bottom panels are imaging YFP fluorescence. (B) Percent of cauline leaves to abscise three days after treatment. Data are mean ± s.e.m.; n = 7 biological replicates (one plant each); letters indicated different statistical quantities t-test <i>P</i> < 0.05. (C) Quantification of HAE-YFP fluorescence two days after infection while all leaves were still attached. Data are mean ± s.e.m.; n = 4 biological replicates (one plant each); letters indicate different statistical quantities t-test <i>P</i> < 0.05. Scale bar is 0.5 mm.</p

DC3000 does not move in cauline leaves from the place of infiltration.

<p>The indicated half of each cauline leaf was infiltrated with DC3000 that express luciferase constitutively driven by the kanamycin promoter. A line was drawn on the leaf with a pen to indicate the border of the infiltrated/not infiltrated. The leaves were cut in half 2 days after infection and imaged with white reflected light (left) and luminescence (right). Four replicates were performed with the same results.</p

Pathogen defense defective mutants are defective in pathogen-triggered cauline leaf abscission.

<p>(A) Percent of cauline leaves that abscised 3 days after infection. Data are mean ± s.e.m.; n = 25 biological replicates (one plant each); t-test versus WT; <i>*P</i> < 0.0001. (B) NahG and <i>pad4</i> floral organ abscission is similar to WT. Red tape is 9.5 mm wide. (C) Relative leaf breakstrength force of WT (Col-0), NahG transgenic plants, and <i>pad4</i> exposed to drought and re-watering conditions. Data are mean ± s.e.m.; n = 12 biological replicates (one plant each); t-test versus WT; <i>*P</i> < 0.05.</p

The bacterial type III-secreted protein AvrRps4 is a bipartite effector

<div><p>Bacterial effector proteins secreted into host plant cells manipulate those cells to the benefit of the pathogen, but effector-triggered immunity (ETI) occurs when effectors are recognized by host resistance proteins. The RPS4/RRS1 pair recognizes the <i>Pseudomonas syringae</i> pv. pisi effector AvrRps4. AvrRps4 is processed <i>in planta</i> into AvrRps4^N (133 amino acids), homologous to the N-termini of other effectors including the native <i>P</i>. <i>syringae</i> pv. tomato strain DC3000 effector HopK1, and AvrRps4^C (88 amino acids). Previous data suggested that AvrRps4^C alone is necessary and sufficient for resistance when overexpressed in heterologous systems. We show that delivering AvrRps4^C from DC3000, but not from a DC3000 <i>hopK1</i>^<i>-</i> strain, triggers resistance in the Arabidopsis accession Col-0. Delivering AvrRps4^C in tandem with AvrRps4^N, or as a chimera with HopK1^N, fully complements AvrRps4-triggered immunity. AvrRps4^N in the absence of AvrRps4^C enhances virulence in Col-0. In addition, AvrRps4^N triggers a hypersensitive response in lettuce that is attenuated by coexpression of AvrRps4^C, further supporting the role of AvrRps4^N as a bona fide effector domain. Based on these results we propose that evolutionarily, fusion of AvrRps4^C to AvrRps4^N may have counteracted recognition of AvrRps4^N, and that the plant <i>RPS4/RRS1</i> resistance gene pair was selected as a countermeasure. We conclude that AvrRps4 represents an unusual chimeric effector, with recognition in Arabidopsis by RPS4/RRS1 requiring the presence of both processed effector moieties.</p></div

Bacteria secreting AvrRps4^N and AvrRps4^C in tandem trigger resistance comparable to wild-type AvrRps4.

<p>Col-0 plants were inoculated with 5x10⁴ cfu/mL suspensions of the indicated DC3000 <i>hopK1</i>^<i>-</i> strains. AvrRps4 variants were delivered from separate broad host range plasmids (top: pML123 constructs; bottom: pVSP61 constructs). Error bars denote standard deviation. Values are averages from four independent experiments with triplicate samples, and error bars denote standard deviation, with letters indicating statistically significant differences (<i>P</i><0.05).</p

HopK1^N/AvrRps4^C chimeras are functional in ETI.

<p>(A) The indicated AvrRps4 and HopK1 constructs were delivered from the <i>P</i>. <i>fluorescens</i> strain EtHAn into Arabidopsis Ws-0. HR response was observed after 24 hours. This experiment was repeated at least twice with similar results. (B) <i>In planta</i> bacterial growth analysis of DC3000 <i>hopK1</i>^<i>-</i> secreting indicated wild-type or chimeric effectors. Ws-0 plants were inoculated with 5x10⁴ cfu/mL suspensions of bacteria. Values are averages from two independent experiments with triplicate samples, and error bars denote standard deviation, with letters indicating statistically significant differences (<i>P</i><0.0001).</p