Characterization of imp-α double and triple mutants

In the previous sections, it has been shown that of the nine Arabidopsis IMP-αs apparently only MOS6 is genetically required for immune responses against virulent H. a.

NOCO2 and Pst DC3000 (ΔAvrPto/AvrPtoB) and growth inhibition caused by auto-activated snc1. However, it is important to note that the loss of MOS6 does not result in complete breakdown of basal resistance and only partial suppression of the snc1-mediated growth phenotype. This could be explained by partially overlapping functions of IMP-α family members. The possibility of redundancy was addressed by crossing imp-α single mutant lines to produce imp-α double and triple mutants that subsequently were characterized. mos6 mutants were combined with mutants of the other IMP-αs except imp-α4 and imp-α9 for which mutant lines were not available when double mutants were generated. Additionally, several other double mutant combinations were generated, e.g.

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the generation of mos6 containing triple mutants, the different mos6 imp-α double mutants were combined.

3.1.4.1 Higher order mutant combinations containing imp-α1 show reduced growth

In order to assess whether the generated imp-α double mutants show any growth phenotypes, plants were grown in parallel on soil under short day and long day conditions and photographed (Figure 3.11). Col-0 plants were used as wild-type controls. Pictures of

Figure 3.11 Phenotypic analysis of imp-α double mutants. Plants were grown in parallel on soil for four weeks under short day (SD) conditions and four or five weeks under long day (LD) conditions.

Scale bar = 1 cm.

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whole rosettes were taken of four-week-old short day and four-week-old long day grown plants. Plants grown in the long day chamber were allowed to bolt and flower and pictures were taken of five-week-old flowering plants. Generally, growth of imp-α double mutants was indistinguishable from Col-0 wild-type plants that were grown under the same conditions. However, double mutants containing the imp-α1 allele were smaller than wild-type plants (Figure 3.11) although imp-α1 single mutants do not show a growth phenotype different from Col-0 (Figure 3.7). This was true for the imp-α1 imp-α2, imp-α1 mos6-1 and imp-α1 mos6-4 double mutant plants. The phenotype was most pronounced in the double mutant of the closely related imp-α1 and imp-α2. For the mos6 imp-α1 combinations this weak reduction was most clearly seen for full grown plants after five weeks growth under LD conditions rather than for rosette sizes. Aside from this no other obvious morphological defects could be observed for any of the imp-α double mutants (Figure 3.11). In addition to double mutants, phenotypes of the generated triple mutant combinations were characterized. Figure 3.12 shows growth phenotypes of the imp-α

Figure 3.12 Phenotypic analysis of imp-α triple mutants. Plants were grown in parallel on soil for four weeks under short day (SD) conditions and four or five weeks under long day (LD) conditions.

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triple mutant lines. snc1 mutants were used as control for reduced growth. The imp-α1 imp-α2 mos6-4 mutant plants were severely stunted in comparison to the imp-α1 imp-α2 and imp-α1 mos6 double mutants and Col-0. In particular, when grown under short day conditions, these plants were even smaller than the growth retarded snc1 plants (Figure 3.12). Aside from this, no other morphological defects could be observed for any of the imp-α triple mutants.

To also assess a possible compensatory up-regulation in the expression of the remaining functional IMPαs in the triple mutants, semi-quantitative RT-PCRs were performed. Figure 3.13 shows the RT-PCR analysis for IMP-αs on cDNA transcribed from RNA extracted from leaves of four-week-old imp-α triple mutant lines. ACTIN1 (ACT1) expression was used as control. This gene expression study shows that there were no significant differences in regard to IMP-α expression in the triple imp-α mutant lines as compared to the wild-type control Col-0.

Figure 3.13 Expression of remaining functional IMP-αs in imp-a triple mutants is not obviously altered. Semi-quantitative RT-PCR analysis of IMP-αs on cDNA transcribed from total RNA extracted from leaves of four-week-old plants of the indicated genotypes. ACTIN1 expression was used as control. When possible, primer combinations were designed to be located in exons and span at least one intron of the tested genes to monitor contamination by genomic DNA based on the size of PCR-fragments. Note that bands corresponding to IMP-α5, IMP-α6, IMP-α7 and IMP-α8 are the same size as Col-0 gDNA bands due to the primer combinations not spanning introns. Numbers of PCR cycles are depicted on the left. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining.

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3.1.4.2 Susceptibility of triple mutant lines containing mos6 is not further increased as compared to the mos6-1 single mutant

This work revealed that loss of MOS6 function in mos6-1, mos6-2 and mos6-4 results in enhanced susceptibility against mildly virulent Pst DC3000 (ΔAvrPto/AvrPtoB) whereas all other imp-α single mutants did show wild-type like susceptibility against this pathogen strain (Figure 3.2, Figure 3.9). To analyze the genetic relationship between MOS6 and other IMP-αs in response to Pst DC3000 (ΔAvrPto/AvrPtoB) the triple mutants were inoculated with this pathogen. The susceptibility of triple mutants containing mos6-4 is similar to that of the mos6-1 single mutant and no additive effects of additional imp-α mutant alleles on susceptibility were observed (Figure 3.14).

Figure 3.14 Triple mutant lines containing mos6 show susceptibility against mildly virulent Pst DC3000 (ΔAvrPto/AvrPtoB) to an extent comparable to mos6-1. Growth of Pst DC3000 (ΔAvrPto/AvrPtoB). The leaves of four-week-old plants of the indicated genotypes were infiltrated with a bacterial suspension of 1 x 10⁵ cfu/mL. To quantify bacterial growth, leaf discs within the inoculated areas were taken immediately (d0) and three days after infection (d3). Bars represent means from two replicate samplings for d0 (white bars) and nine replicate samplings for d₃ (black bars). Error bars show standard deviation and asterisks indicate statistical significance (t-test for pairwise comparison of wild-type and mutants, * p<0.05,

** p<0.01). snc1 and eds1 (Col eds1-2) are resistant and susceptible controls, respectively. Cfu = colony-forming units. All experiments were repeated twice with similar results.

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Together, the analyses of imp-α triple mutant combinations show that IMP-α1, IMP-α2 and MOS6/IMP-α3 have partially redundant functions that are important for regular plant growth and development. In contrast, MOS6 appears to be selectively required for maintaining the basal resistance layer against Pst DC3000 (ΔAvrPto/AvrPtoB) as the susceptibility of the triple mutants containing mos6-4 was not further increased as compared to the mos6-1 single mutant.

3.2 Identification and characterization of defense-related cargo proteins and