β-carbonic anhydrases play a role in salicylic acid perception in Arabidopsis

The plant hormone salicylic acid (SA) is required for defense responses. NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) and NON RECOGNITION OF BTH-4 (NRB4) are required for the response to SA in Arabidopsis (Arabidopsis thaliana). Here, we isolated several interactors of NRB4 using yeast two-hybrid assays. Two of these interactors, βCA1 and βCA2, are β-carbonic anhydrase family proteins. Since double mutant βca1 βca2 plants did not show any obvious phenotype, we investigated other βCAs and found that NRB4 also interacts with βCA3 and βCA4. Moreover, several βCAs interacted with NPR1 in yeast, including one that interacted in a SA-dependent manner. This interaction was abolished in loss-of-function alleles of NPR1. Interactions between βCAs and both NRB4 and NPR1 were also detected in planta, with evidence for a triple interaction, NRB4- βCA1-NPR1. The quintuple mutant βca1 βca2 βca3 βca4 βca6 showed partial insensitivity to SA. These findings suggest that one of the functions of carbonic anhydrases is to modulate the perception of SA in plants.Facultad de Ciencias Exacta

Alteraciones en la percepción del ácido salicílico por mutagénesis en levadura

En nuestro laboratorio trabajamos en la percepción del ácido salicílico (SA) por la planta Arabidopsis thaliana. El SA es la primera molécula a la que se atribuyó un papel importante en la intermediación de la transducción de la señal patogénica. El incremento en los niveles endógenos de SA y de sus conjugados en plantas infectadas coincide con la activación de genes que codifican para proteínas PR (Relacionadas con Patogénesis) y con el establecimiento de la resistencia en la planta. De este modo, las plantas que no perciben SA son más susceptibles a algunos patógenos, mientras que la aplicación del SA aumenta la resistencia de las plantas. La cascada de señalización mediada por SA está constituida por un conjunto de proteínas que juegan un papel importante en el establecimiento y prolongación de la respuesta de defensa (SAR; Resistencia Sistémica Adquirida). Entre ellas se han identificado NPR1 (No expresor de genes PR) y βCAs (Anhidrasa Carbónica). NPR1 codifica para un regulador positivo de SAR, actuando corriente abajo del SA. La función de este gen es conservada en numerosas especies de plantas y los mutantes deficientes son sensibles al ataque por patógenos. Las CAs son metaloenzimas que catalizan la conversión rápida de dióxido de carbono y agua a bicarbonato y protones. El centro activo de la mayoría de las CAs contiene un ión de zinc. En plantas se han identificado CAs de tipo α, β y γ que participan en la fijación de carbono del CO2 en diferentes partes de la célula. Concretamente, ciertas βCAs han sido descritas como proteínas de unión al SA (SABP) y su papel en la fotosíntesis está cuestionado. Trabajos previos en nuestro laboratorio mediante un rastreo en el sistema de doble híbrido de levadura (Y2H), han permitido identificar que las proteínas NPR1 y CA1 interaccionan sólo en presencia de SA. El trabajo a desarrollar por el alumno como Trabajo de Fin de Máster consiste en el estudio de dicha interacción (aumentándola o disminuyéndola) por diferentes aproximaciones. La ventaja de utilizar levadura es que es fácil seleccionar aumentos o disminuciones de la interacción alterando la composición del medio. Esta aproximación consiste en llevar a cabo mutagénesis de NPR1 y CA1 por PCR mediante el sistema RCA (Rolling Circle Amplification) que emplea la ADN polimerasa del fago Φ29. Una vez mutagenizadas estas proteínas, el alumno procederá a rastrear aquellas mutaciones que sean de mayor interés, buscando, preferentemente, mutaciones que generen (1) interacción independiente de SA, (2) mayor afinidad por el SA, y (3) no interacción. A continuación, una vez identificadas las proteínas mutagenizadas de interés, se procederá a testar su (1) capacidad de unión a SA y (2) su actividad CA en el caso de CA1. En el caso de las mutaciones de NPR1, (1) se testarán las interacciones con otras CAs que normalmente interaccionan con NPR1. En función de la consecución de los objetivos anteriores, cabe la posibilidad de abordar nuevos propósitos tales como (1) la construcción de una genoteca de ADN copia de Arabidopsis en pARC352 para la generación de un triple híbrido de levadura que contenga NPR1+CA1 junto con un tercer miembro que interfiera en la interacción, bien de forma positiva o negativa y (2) llevar a cabo un rastreo en levadura mediante genética química con el propósito de identificar nuevos compuestos químicos que interfieran en la interacción NPR1+CA1 de forma antagonista y también agonista.Lamilla Monje, JR. (2017). Alteraciones en la percepción del ácido salicílico por mutagénesis en levadura. http://hdl.handle.net/10251/79904TFG

NPR1 paralogs of Arabidopsis and their role in salicylic acid perception.

Salicylic acid (SA) is responsible for certain plant defence responses and NON EXPRESSER OF PATHOGENESIS RELATED 1 (NPR1) is the master regulator of SA perception. In Arabidopsis thaliana there are five paralogs of NPR1. In this work we tested the role of these paralogs in SA perception by generating combinations of mutants and transgenics. NPR2 was the only paralog able to partially complement an npr1 mutant. The null npr2 reduces SA perception in combination with npr1 or other paralogs. NPR2 and NPR1 interacted in all the conditions tested, and NPR2 also interacted with other SA-related proteins as NPR1 does. The remaining paralogs behaved differently in SA perception, depending on the genetic background, and the expression of some of the genes induced by SA in an npr1 background was affected by the presence of the paralogs. NPR2 fits all the requirements of an SA receptor while the remaining paralogs also work as SA receptors with a strong hierarchy. According to the data presented here, the closer the gene is to NPR1, the more relevant its role in SA perception

NRB4 interacts with βCA1f and βCA2f.

<p>(A) Yeast cells transformed with the indicated plasmids and inserts were grown on three different sets of plates. The first set contained minimal medium supplemented with histidine (+His), the second contained the same minimal medium with no histidine (-His), 100 μM salicylic acid (SA), and 100 mM 3-Amino-1,2,4-triazole (3AT), and the third lacked histidine (-His) and contained 100 μM 4-hydroxybenzoic acid (4HBA) and 100 mM 3AT. The first three rows indicate that NRB4, βCA1f, or βCA2f alone do not allow the yeast to grow in medium lacking histidine. The growth of yeast in the remaining two rows on -His plates indicates that NRB4 interacts with βCA1f and βCA2f. Note that the presence of SA or 4HBA does not affect the interaction. (B) βCA1f and βCA2f interact with the point mutations of NRB4 <i>in planta</i>. The interactions between βCA1 and βCA2 with nrb4-2 and nrb4-3 were tested as mentioned in “A”. (C) βCA1f interacts with the KIX domain of NRB4. The interaction between βCA1f and three different constructs of NRB4 was tested as mentioned in “A”, except that the -His plates did not contain 3AT. (D) βCA2f interacts with the KIX domain of NRB4. The interaction between βCA2f and three different constructs of NRB4 was tested as mentioned in “C”, with the controls shown in “C”. (E) Diagram of the NRB4 constructs used in the previous panels. The names of the domains are under the wild type NRB4. The red arrows indicate the point mutations found [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.ref020" target="_blank">20</a>]. (F) CA activity in different genotypes and under different treatments. Two weeks old Col-0 plants were treated with mock solution, 1 mM SA, or 350 μM benzothiadiazole (BTH), and the samples were frozen one day later. Similarly, Col-0, <i>NahG eds5 sid2</i>, <i>npr1</i>-1, and <i>nrb4</i>-2 plants were inoculated with <i>Pseudomonas syringae</i> pv. <i>tomato</i> isolate DC3000 (<i>Pto</i>) at an OD₆₀₀ of 0.1, and the samples were frozen one day later. The samples (three repeats of approximately 100 mg each) were ground and CA activity measured based on the change in color of bromothymol blue following the change in pH. In all figures where numerical information is presented, the data represent the average values, with error bars showing standard deviation. The letters above the bars indicate different homogeneous groups with statistically significant differences (Fisher’s LSD Test, P < 0.05). All experiments were repeated at least three times with similar results.</p

Quantification of the βCA1f-NPR1 interaction; other βCAs also interact with NPR1.

<p>(A) Dose response to SA in the interaction between βCA1f and NPR1. Yeast containing both cDNAs was grown in the presence of SA, and β-galactosidase activity was then measured, since the interaction between the two cDNAs leads to the expression of this enzyme. (B) The βCA1f-NPR1 interaction requires a functional analog of SA; 3HBA represents 3-hydroxybenzoic acid, and the interaction was quantified as described in “A”. (C) The inactive analogs competed poorly with SA, while active analogs showed an additive effect. The interaction was quantified as described in “A”. In panels “B” and “C”, 100 μM of each chemical was added to the medium. (D) Yeast three hybrid. Several versions of NRB4, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.g001" target="_blank">Fig 1</a>, were cloned in a third plasmid and introduced into yeast with βCA1f and NPR1 to show a triple interaction. (E) Five additional βCAs were tested as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.g001" target="_blank">Fig 1C</a>. (F) Six additional βCAs were tested as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.g001" target="_blank">Fig 1C</a>. In total, βCA1.6, βCA2.2, βCA2.7, βCA2.8, βCA3.1, βCA3.2, and βCA4.1 also interacted with NPR1, although less strongly and not depending on SA, as is the case with βCA1f.</p

SA perception phenotypes of the βCA T-DNAs.

<p>(A) CA activity in plants with a single T-DNA insertion in the βCA genes and combinations of these mutants. In the case of <i>βca5</i>, the homozygous plant was sterile (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.s011" target="_blank">S11 Fig</a>), and the progeny of a heterozygous plant were used. The activity was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.g001" target="_blank">Fig 1F</a>; <i>4xβca</i> represents <i>βca1 βca2 βca4 βca6</i>, while <i>5xβca</i> represents <i>βca1 βca2 βca3 βca4 βca6</i>. (B) Combinations of T-DNA insertions reduce SA and BTH perception. 14-day-old plants were treated with 500 μM SA, 350 μM BTH, or mock solution. One day later, the plants were inoculated with <i>Pseudomonas syringae</i> pv. <i>tomato</i> isolate DC3000 (<i>Pto</i>) at an OD₆₀₀ of 0.1. Three days after inoculation, <i>Pto</i> growth was evaluated as the logarithm of colony forming units (cfu) per plant. The remaining <i>βca</i> genotypes are showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.s012" target="_blank">S12 Fig</a>. (C) Decrease in effector-triggered immunity. The indicated genotypes were inoculated as in “B” with different <i>Pto</i> strains containing the indicated effectors. The numbers after the letters indicate that these are independent experiments and only data with the same number can be compared. The complete set of experiments is showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.s012" target="_blank">S12 Fig</a>. (D) eds-like phenotype. Seven-week-old plants were hand infiltrated with <i>Pto</i> at an OD₆₀₀ of 10⁻⁴. Three days after inoculation, <i>Pto</i> growth was evaluated as the logarithm of cfus per g of fresh weight. (E) Decrease in the toxic effect of SA. The <i>βca</i> mutants and the controls were grown on MS plates supplied with 0, 200, and 300 μM SA (photographs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.s012" target="_blank">S12 Fig</a>), and the chlorophyll contents of the plants were measured as an indication of the response to SA (30 plants in three groups of 10). (F) The <i>βca</i> mutants accumulate more SA than wild type. The SA levels (both free and total) were measured three days after mock or <i>Pto</i> inoculation as in “B”, with samples of 15 plants in three groups of five.</p

βCA1f interacts with NPR1 in the presence of SA.

<p>(A) The interactions between βCA1f and several proteins related to SA perception were tested as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.g001" target="_blank">Fig 1C</a>. βCA1f interacts with NPR1 in a SA-dependent manner, while it interacts with TGA2 regardless of SA. (B) βCA1f did not interact with any of the six <i>npr1</i> alleles tested. These alleles are point mutations of NPR1 found <i>in planta</i>, and they produce stable protein. (C) βCA1f interacted with two NPR1 point mutations that do not alter NPR1 function [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.ref056" target="_blank">56</a>]. (D) Mutations that disrupt CA activity affect the interaction between βCA1f and NPR1. Two mutations that produce stable pea CA with no activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.ref058" target="_blank">58</a>] were re-created in βCA1f. (E) The same two mutations in βCA1f also affected the interaction between βCA1f and NRB4.</p

βCAs interact <i>in planta</i> with NRB4 and NPR1.

<p>Interaction of NRB4 with βCAs. (A) Bimolecular fluorescence complementation (BiFC) showing a negative interaction (as a control), and (B) positive interaction (detectable GFP). In total, four βCAs interacted with NRB4. Interaction of NPR1 with βCAs. Similarly, (C) shows a lack of interaction, while (D) shows a positive interaction. In total, six βCAs interacted with NPR1. The complete series of images can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.s007" target="_blank">S7 Fig</a>. The bars represent 20 μm. (E) <i>GFP</i>-<i>NPR1</i> and different <i>MBP-βCAs</i> were transiently expressed in <i>N</i>. <i>benthamiana</i> by agroinfiltration and pulled-down with amylose resin. The panel shows the eluted fraction from the resin, detecting GFP-NPR1 (upper) and MBP-βCAs (lower) by immunoblot analysis with the indicated antibodies. (F) The same experiment after 1 mM SA treatment. Additional controls are showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.s009" target="_blank">S9 Fig</a>.</p

Role of βCAs in SA perception.

<p>(A) SA binding of half of the cloned βCAs. Purified recombinant proteins were incubated with 4-AzSA, followed by UV light treatment. 4-AzSA-cross-linked proteins were detected by immunoblot analysis with antibody against SA. The first line corresponds to the negative control, where UV light was omitted. (B) SA binding of the remaining cloned βCAs. (C) Competition for SA binding between βCA1f and NPR1. Similar to “A”, purified recombinant βCA1f and NPR1 proteins were decorated with anti-SA alone or combined. An immunoblot with anti-MBP is shown as a control for protein input. (D) Changes in the localization of βCA1.3-GFP upon SA treatment. Stable transgenic Arabidopsis plants were observed under a confocal microscope one day after treatment. (E) Relative abundance of βCA1f and NPR1 when expressed from the same promoter. Stable transgenic Arabidopsis plants harboring GFP, GFP-βCA1f, and GFP-NPR1 were subject to immunoblot analysis using an anti-GFP antibody. The letters indicate independent lines. In the case of βCA1f, the progeny of a heterozygous plant were analyzed, since no homozygous plant was identified. In the case of NPR1, line a is in the <i>npr1</i>-70 background [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.ref056" target="_blank">56</a>], and line b is in the <i>npr1</i>-1 background (this work). A <i>NPR1-GFP</i> line in the wild-type background [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.ref057" target="_blank">57</a>] was also tested. Except for the <i>NPR1-GFP</i> line, the remaining constructs are in the same plasmid backbone, pMDC43. Additional controls for this figure are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.s015" target="_blank">S15 Fig</a>.</p

CA activity.

<p>CA activity in yeast was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#pone.0181820.g001" target="_blank">Fig 1F</a>, but with 1 mL of culture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181820#sec002" target="_blank">Methods</a>). (A) Activity of the cloned βCAs in pDEST22 when pDEST32 is empty. <i>βCA1f</i>E represents <i>βCA1f</i>E110A, and <i>βCA1f</i>C represents <i>βCA1f</i>C129S. (B) Activity of βCAs in pDEST22 that interact with NRB4 when <i>NRB4</i> is present in pDEST32. (C) Activity of βCAs in pDEST32 that interact with NPR1 when pDEST22 is empty. (D) Activity of βCAs in pDEST32 that interact with NPR1 when <i>NPR1</i> is present in pDEST22. (E) Activity of βCA1f in pDEST32 when some NPR1 variations are cloned in pDEST22. <i>NPR1</i>C8 represents <i>NPR1</i>C82A, and <i>NPR1</i>C21 represents <i>NPR1</i>C216A. (F) Activity of βCAs in pDEST22 that interact with NRB4 when <i>NRB4</i> is present in pDEST32, and in the presence of 100 μM SA, compared to “B”. (G) Activity of <i>βCA1f</i>E110A cloned in pDEST32, when NPR1 is cloned in pDEST22, and in the presence of SA 100 μM, compared to “D”. (H) Activity of βCA1f in pDEST32 when some NPR1 variations are cloned in pDEST22, and in the presence of SA, compared to “E”.</p