FaNPR3 Members of the NPR1-like Gene Family Negatively Modulate Strawberry Fruit Resistance against Colletotrichum acutatum
<p>Expression pattern of <span class="html-italic">FaNPR3</span> genes in strawberry fruit after agroinfiltration with pFRN::FaNPR3<span class="html-italic">all</span>.RNAi construct. (<b>A</b>) Gene expression of <span class="html-italic">FaNPR3.1</span>. (<b>B</b>) Gene expression of <span class="html-italic">FaNPR3.2</span>. (<b>C</b>) Gene expression of <span class="html-italic">FaNPR3.3</span>. qRT-PCR analysis was accomplished in agroinfiltrated strawberry fruit before (0 and 2 days) and after (3, 5, and 7 days) <span class="html-italic">C. acutatum</span> inoculation (<span class="html-italic">Ca</span>). The time scale refers to days after agroinfiltration (dai). For all the genes, the arrow and horizontal line indicate the time of <span class="html-italic">Ca</span> inoculation. The black and grey columns show pFRN (control) and pFRN::FaNPR3<span class="html-italic">all</span>.RNAi (silencing) agroinfiltrations, respectively. Data from all time points are referred to data in time zero, represented as 1 (white column). Bars, mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span> < 0.05) in HSD Tukey’s post hoc test.</p> "> Figure 2
<p>Expression pattern of <span class="html-italic">FaNPR3</span> genes in strawberry fruit after agroinfiltration with pB7GWIWG2::FaNPR32.RNAi. (<b>A</b>) Gene expression of <span class="html-italic">FaNPR3.1</span>. (<b>B</b>) Gene expression of <span class="html-italic">FaNPR3.2</span>. (<b>C</b>) Gene expression of <span class="html-italic">FaNPR3.3</span>. qRT-PCR analysis was accomplished in agroinfiltrated strawberry fruit before (0 and 2 days) and after (3, 5, and 7 days) <span class="html-italic">C. acutatum</span> inoculation (<span class="html-italic">Ca</span>). The time scale refers to days after agroinfiltration (dai). For all the genes, the arrow and the horizontal line indicate the time of <span class="html-italic">Ca</span> inoculation. The black and grey columns show pB7GWIWG2 and pB7GWIWG2::FaNPR32.RNAi agroinfiltrations, respectively. Data from all time points are referred to data in time zero, represented as 1 (white column). Bars, mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span> < 0.05) in HSD Tukey’s post hoc test.</p> "> Figure 3
<p>Silencing effect of pFRN::FaNPR3<span class="html-italic">all</span>.RNAi and ectopic overexpression of <span class="html-italic">FaNPR3.1</span> in strawberry fruit after <span class="html-italic">C. acutatum</span> infection. (<b>A</b>,<b>B</b>) Upper panels: external surface disease symptoms on the two agroinfiltrated opposite halves of the same fruit, after silencing and overexpression, respectively. (<b>A</b>,<b>B</b>) Lower panels: internal tissue damage of the same fruit shown in the corresponding upper panels. pFRN::FaNPR3<span class="html-italic">all</span>.RNAi and pFRN, silencing construct and its corresponding empty vector, as control. pK7::FaNPR31.OE and pK7, overexpression construct and its corresponding empty vector, as control. A relevant fruit is shown for each condition, as an example. (<b>C</b>) Statistical analysis of internal tissue damage ratio of the two opposite halves of the same fruit, according to the 1 to 5 severity scale; striped and plain blue bars, pFRN/pFRN and pFRN/pFRN::FaNPR31-RNAi agroinfiltrated values, respectively; striped and plain red bars, pK7/pK7 and pK7/pK7::FaNPR31-OE agroinfiltrated values, respectively. Data correspond to mean ± SD. Within each bar, means with different letters are significantly different by LSD test at <span class="html-italic">p</span> < 0.05. A ratio value of 1 indicates no differences between the opposite halves of the same fruit.</p> "> Figure 4
<p>Silencing effect of pB7GWIWG2::FaNPR32.RNAi and ectopic overexpression of <span class="html-italic">FaNPR3.2</span> in strawberry fruit after <span class="html-italic">C. acutatum</span> infection. (<b>A</b>,<b>B</b>) Upper panels: external surface disease symptoms on the two agroinfiltrated opposite halves of the same fruit, after silencing and overexpression, respectively. (<b>A</b>,<b>B</b>) Lower panels: internal tissue damage of the same fruit shown in the corresponding upper panels. pB7GWIWG2::FaNPR32.RNAi and pB7GWIWG2, silencing construct and its corresponding empty vector, as control. pB7WG2::FaNPR32.OE and pB7WG2, overexpression construct and its corresponding empty vector, as control. A relevant fruit is shown for each condition, as an example. (<b>C</b>) Statistical analysis of internal tissue damage ratio of the two opposite halves of the same fruit, according to the 1 to 5 severity scale; striped and plain blue bars, pB7GWIWG2/pB7GWIWG2 and pB7GWIWG2/pB7GWIWG2::FaNPR32-RNAi agroinfiltrated values, respectively; striped and plain red bars, pB7WG2/pB7WG2 and pB7WG2/pB7WG2::FaNPR32-OE agroinfiltrated values, respectively. Data correspond to mean ± SD. Within each bar, means with different letters are significantly different by LSD test at <span class="html-italic">p</span> < 0.05. A ratio value of 1 indicates no differences between opposite halves of the same fruit.</p> "> Figure 5
<p>Silencing effect of <span class="html-italic">FaNPR3</span> members on the expression of <span class="html-italic">FaWRKY19</span> and <span class="html-italic">FaWRKY24</span> genes in strawberry fruit after <span class="html-italic">C. acutatum</span> infection. (<b>A</b>) Silencing effect of pFRN::FaNPR3<span class="html-italic">all</span>.RNAi. (<b>B</b>) Silencing effect pB7GWIWG2::FaNPR32.RNAi. Black columns, the expression of <span class="html-italic">FaWRKY19</span> and <span class="html-italic">FaWRKY24</span> genes in half fruit agroinfiltrated with pFRN (<b>A</b>) or pB7GWIWG2 (<b>B</b>) control vectors. Grey columns, the expression of <span class="html-italic">FaWRKY19</span> and <span class="html-italic">FaWRKY24</span> genes in half fruit agroinfiltrated with pFRN::FaNPR3<span class="html-italic">all</span>.RNAi (<b>A</b>) or pB7GWIWG2::FaNPR32.RNAi (<b>B</b>). qRT-PCR analysis was accomplished in agroinfiltrated strawberry fruit before (0 and 2 days) and after (3, 5, and 7 days) <span class="html-italic">C. acutatum</span> inoculation (<span class="html-italic">Ca</span>). The time scale refers to days after agroinfiltration (dai). Arrow and horizontal line indicate the time of <span class="html-italic">Ca</span> inoculation. Data from all time points are referred to data in time zero, represented as 1 (white column). Bars, mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span> < 0.05) in HSD Tukey’s post hoc test.</p> "> Figure 6
<p>Characterization of Arabidopsis lines. (<b>A</b>) <span class="html-italic">FaNPR3.2</span> expression in Arabidopsis WT-, <span class="html-italic">npr3npr4</span> mutant-, and <span class="html-italic">FaNPR3.2</span>-overexpressing lines <span class="html-italic">npr3npr4</span>::FaNPR32 and WT::FaNPR32 (black, grey, white and striped bars, respectively). Transcript accumulation was monitored by qRT-PCR in non-infected plants as described in the <a href="#sec4-plants-13-02261" class="html-sec">Section 4</a>. Expression levels were normalized with respect to the internal control <span class="html-italic">ACTIN2</span> and displayed relatively to the threshold value of the wild-type (no expression of <span class="html-italic">FaNPR3.2</span>) that was given a value of 1 for convenience. Note that no amplification of <span class="html-italic">FaNPR3.2</span> strawberry gene is detected in neither WT nor double mutant <span class="html-italic">npr3npr4</span> using the specific primers (<a href="#app1-plants-13-02261" class="html-app">Supplementary Materials Table S1</a>). Bars represent the mean levels of transcript quantified from three independent biological experiments (±SD). (<b>B</b>–<b>D</b>) Disease resistance phenotype of Arabidopsis lines upon <span class="html-italic">Pst</span> inoculation. (<b>B</b>) Symptoms development on leaves 3, 5, and 7 days post-inoculation (dpi) (10<sup>6</sup> CFU mL<sup>−1</sup>); (<b>C</b>) trypan blue staining for the detection of cell death 1 dpi (10<sup>5</sup> CFU mL<sup>−1</sup>); (<b>D</b>) in planta bacterial growth monitored 3, 5, and 7 days post-inoculation (10<sup>6</sup> CFU mL<sup>−1</sup>). CFU, colony-forming units. Statistically significant differences are labelled by letters (one-way ANOVA, Tukey’s multiple comparisons test, <span class="html-italic">p</span> < 0.05). <span class="html-italic">Pst</span> was pressure infiltrated into fully expanded mature leaves of 4–5-week-old Arabidopsis plants. As control, leaves were infiltrated with 10 mM MgCl<sub>2</sub>. The whole experiment was performed three times with similar results.</p> "> Figure 7
<p>Expression of the defense-related genes <span class="html-italic">PR1</span>, <span class="html-italic">PR2</span>, and <span class="html-italic">PR5</span> in Arabidopsis lines. Relative expression level was monitored by qRT-PCR in control (<b>A</b>) and infected plants 1 and 2 days post-inoculation (dpi) with <span class="html-italic">Pst</span> (10<sup>6</sup> CFU mL<sup>−1</sup>) (<b>B</b>), in WT, WT::FaNPR32, double mutant <span class="html-italic">npr3npr4</span>, and <span class="html-italic">npr3npr4</span>::FaNPR32 (black, grey, white and striped bars, respectively). Expression levels were normalized with respect to the internal control <span class="html-italic">ACTIN2</span> and displayed relative to the expression in mock-treated wild-type samples (<b>A</b>) or to the expression in mock-treated samples of each line (<b>B</b>) that were given a value of 1. Bars refer to mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span> < 0.05) in HSD Tukey’s post hoc test.</p> "> Figure 8
<p>Expression of the endogenous <span class="html-italic">AtNPR1</span>, <span class="html-italic">AtNPR3</span>, and <span class="html-italic">AtNPR4</span> genes in Arabidopsis. Relative expression level was monitored by qRT-PCR in control (<b>A</b>) and infected plants 1 and 2 days post-inoculation (dpi) with <span class="html-italic">Pst</span> (10<sup>6</sup> CFU mL<sup>−1</sup>) (<b>B</b>), in WT, WT::FaNPR32, double mutant <span class="html-italic">npr3npr4</span>, and <span class="html-italic">npr3npr4</span>::FaNPR32 (black, grey, white and striped bars, respectively). Expression levels were normalized with respect to the internal control <span class="html-italic">ACTIN2</span> and displayed relative to the expression in mock-treated wild-type samples (<b>A</b>) or to the expression in mock-treated samples of each line (<b>B</b>) that were given a value of 1. Bars refer to mean ± standard error. Note the different scales in the relative-expression-level axis. Statistical significance was determined by one-way ANOVA. Letters indicate significant differences (<span class="html-italic">p</span> < 0.05) in HSD Tukey’s post hoc test. Note that the absence of transcript for <span class="html-italic">AtNPR3</span> and <span class="html-italic">AtNPR4</span> in the overexpressing lines in the double mutant <span class="html-italic">npr3npr4</span> background. It proves that Arabidopsis primers do not amplify strawberry orthologous genes.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Expression Pattern of FaNPR Gene Family after Transient Silencing and Overexpression of the FaNPR3 Members in Strawberry Fruit
2.2. The Silencing of FaNPR3 Genes in Strawberry Fruit Reduced Fruit Tissue Damage after C. acutatum Inoculation
2.3. Analysis of Defense-Related Genes in Strawberry Fruit Silenced in FaNPR3 Genes
2.4. The Strawberry FaNPR3.2 Gene Complements the Arabidopsis Atnpr3npr4 Double Mutant Disease Resistance Phenotype
2.5. Changes in the Expression Profile of Defense-Related Genes in Arabidopsis
3. Discussion
3.1. Members of the FaNPR3 Clade Negatively Modulate Strawberry Fruit Resistance against Colletotrichum acutatum
3.2. FaNPR3.2 Negatively Modulates Resistance in Arabidopsis
3.3. Silencing of FaNPR3 Members in Strawberry Fruit Downregulates FaWRKY19 and FaWRKY24 Gene Expression
3.4. Resistance to Pseudomonas syringae in Arabidopsis Plants Overexpressing FaNPR3.2 Is Uncoupled from PRs Gene Expression
4. Materials and Methods
4.1. Biological Material, Growing Conditions, and Pathogen and Elicitor Treatments
4.2. Plasmid Construction for Silencing and Overexpressing FaNPR3 Genes
4.3. Agroinfiltration of Strawberry Fruit and Experimental Design
4.4. Stable Transformation of Arabidopsis Plants
4.5. Arabidopsis Infection Assay with Pseudomonas syringae
4.6. RNA Extraction and Real-Time qPCR
4.7. Assessment and Statistical Analysis after Pathogen Infection
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Súnico, V.; Higuera, J.J.; Amil-Ruiz, F.; Arjona-Girona, I.; López-Herrera, C.J.; Muñoz-Blanco, J.; Maldonado-Alconada, A.M.; Caballero, J.L. FaNPR3 Members of the NPR1-like Gene Family Negatively Modulate Strawberry Fruit Resistance against Colletotrichum acutatum. Plants 2024, 13, 2261. https://doi.org/10.3390/plants13162261
Súnico V, Higuera JJ, Amil-Ruiz F, Arjona-Girona I, López-Herrera CJ, Muñoz-Blanco J, Maldonado-Alconada AM, Caballero JL. FaNPR3 Members of the NPR1-like Gene Family Negatively Modulate Strawberry Fruit Resistance against Colletotrichum acutatum. Plants. 2024; 13(16):2261. https://doi.org/10.3390/plants13162261
Chicago/Turabian StyleSúnico, Victoria, José Javier Higuera, Francisco Amil-Ruiz, Isabel Arjona-Girona, Carlos J. López-Herrera, Juan Muñoz-Blanco, Ana María Maldonado-Alconada, and José L. Caballero. 2024. "FaNPR3 Members of the NPR1-like Gene Family Negatively Modulate Strawberry Fruit Resistance against Colletotrichum acutatum" Plants 13, no. 16: 2261. https://doi.org/10.3390/plants13162261