Brush and Spray: A high throughput systemic acquired resistance assay suitable for large-scale genetic screening

: Systemic acquired resistance (SAR) is a defense mechanism induced in the distal parts of plants after primary infection. It confers long-lasting protection against a broad spectrum of microbial pathogens. Lack of high-throughput assays has hampered forward genetic analysis of SAR. Here we report the development of an easy and efficient assay for SAR and its application in a forward genetic screen for SAR-deficient mutants. Using the new assay for SAR, we identified six fmo1 , four ald1 , three sid2 , one pad4 and one pbs3 alleles as well as a gain-of-function mutant of CAMTA3 designated camta3-3D. Like transgenic plants over-expressing CAMTA3 , camta3-3D mutant plants exhibit compromised SAR and enhanced susceptibility to virulent pathogens, suggesting that CAMTA3 is a critical regulator of both basal resistance and SAR.


INTRODUCTION
Systemic acquired resistance (SAR) is a general defense response that develops in the distal, uninfected parts of plants after local infection. The term SAR was coined by Ross in the 1960s from his studies on the interactions between tobacco and tobacco mosaic virus (TMV) (Ross, 1961). Once SAR is established, it is believed to be long-lasting and effective against a broad-spectrum of pathogens including viruses, bacteria and fungi. Traditionally, induction of SAR is achieved by local infection using an avirulent (Avr) pathogen that can trigger hypersensitive responses (HR).
However, one study suggests that tissue necrosis at the site of infection is not required for SAR activation (Mishina and Zeier, 2007).
Forward genetic analysis on SAR during the early 1990s focused on components downstream of SA. NPR1 (Nonexpressor of Pathogenesis Related genes 1; also named NIM1 or SAI1) was found to be required for SA-induced PR gene expression and pathogen resistance (Cao et al., 1994;Ryals et al., 1997;Shah et al., 1997). A In traditional Arabidopsis SAR assays, local leaves are first infiltrated with avirulent bacterial pathogens and the systemic leaves are later infiltrated with virulent bacterial pathogens. Induction of SAR is determined by quantifying bacterial growth in the systemic leaves (Cameron et al., 1994). The procedure is tedious and not suitable for large-scale screening of SAR-deficient mutants. We previously developed an assay that significantly improved the efficiency of SAR analysis (Zhang et al., 2010 Figure 1A). On the other hand, when virulent P.s.m.ES4326 was brushed on the leaves, SAR against H.a. Noco2 was consistently induced at doses higher than OD 600 = 0.1 ( Figure 1B). Thus induction of SAR in subsequent studies was carried out by brushing local leaves with P.s.m.ES4326 at OD 600 =0.4.

Identification of SAR-deficient mutants
Because the H.a. Noco2-based method is easy to perform and gives consistent and highly reproducible results, we decided to use it for forward genetic screening to search for SAR deficient mutants. About 45,000 M2 plants from an EMS-mutagenized mutant population in the Col-0 background were analyzed for loss of SAR against H.a. Noco2. About 50 mutants with compromised SAR were obtained.
Sixteen mutants with severe SAR deficiency were chosen for initial analysis. Crude mapping and subsequent targeted sequencing analysis revealed that among them, there were six fmo1 alleles, four ald1 alleles, three sid2 alleles, one pad4 (phytoalexin deficient 4) allele and one pbs3 allele (Table 1). As shown in Figure 3, SAR was severely compromised in these mutants. Only one of the mutants was mapped to a region without known SAR mutants, and we designated this mutant sard3 (SAR-deficient 3).

Characterization of sard3
As shown in Figure 4A, sard3 exhibited strong SAR defects. Interestingly, the enhanced disease susceptibility indicates that SARD3 also plays an essential role in the regulation of basal resistance against bacterial pathogens.

sard3 carries a gain-of-function mutation in CAMTA3/ SR1
To map the sard3 mutation, we crossed sard3 ( semi-dominant and it displays phenotypes opposite of the knockout mutants of CAMTA3, it probably carries a gain-of-function mutation in CAMTA3, which leads to enhanced disease susceptibility in sard3. To test whether the mutation in CAMTA3 is responsible for the SAR deficiency in sard3, we transformed the mutant camta3 gene under the control of its native promoter into wild-type background. About three quarters of the transgenic lines exhibited compromised SAR and we analyzed two representative transgenic lines in detail. As shown in Figure 5C, SAR was compromised in both line T-1 and T-2, but the SAR deficiency phenotype was more severe in line T-1 than line T-2. Real-time RT-PCR analysis showed that line T-1 and T-2 expressed the camta3 transgene at different levels, with higher expression in line T-1 than in line T-2 ( Figure 5D). We also tested growth of P.s.t. DC3000 in the transgenic lines. As shown in Figure 5E, both line T-1 and line T-2 exhibited enhanced disease susceptibility, but line T-1 was much more susceptible to the pathogen than line T-2. These data confirm that sard3 carries a gain-of-function mutation of CAMTA3. To be consistent with the literature, we renamed sard3 to camta3-3D.

Over-expression of CAMTA3 leads to compromised SAR and local resistance
To test whether over-expression of CAMTA3 also results in SAR deficiency, we expressed CAMTA3 under the control of the cauliflower mosaic virus 35S promoter in wild type Col-0 background. Two independent transgenic lines that express high levels of CAMTA3 ( Figure 6A) were analyzed in detail. As shown in Figure 6B, SAR was compromised in both lines and the increased SAR deficiency associated with increased expression of CAMTA3. When these two transgenic lines were challenged with P.s.t. DC3000, both lines supported higher bacterial growth than wild type plants ( Figure 6C). These data further support that CAMTA3 is a negative regulator of both basal defense and SAR. assay also allowed us to map the mutations using F2 progeny from the cross between the SAR mutants and Ler-rpp5.

DISCUSSION
In the screen for SAR-deficient mutants, we identified a gain-of-function allele of CAMTA3 in addition to mutations in genes known to be required for SAR. The sequence of the IQ motif ( Figure S1). It may cause a conformational change of the protein leading to enhanced activity in repressing defense gene expression.
Previously it was shown that CAMTA3 binds to the promoter of EDS1 and negatively regulates its expression. Since over-expression of EDS1 alone is not sufficient to constitutively activate plant immunity (Garcia et al.), there should be additional genes that are regulated by CAMTA3 and contribute to the activation of defense responses in the CAMTA3 knockout mutants. Our real-time RT-PCR analysis showed that the expression of both EDS1 is not affected by the camta3-3D mutation ( Figure S2), suggesting that loss of SAR and enhanced susceptibility to P.s.m.
ES4326 and P.s.t. Dc3000 in camta3-3D are caused by misregulation of other genes.
Future identification of these defense regulators will help us better understand how CAMTA3 negatively regulates basal resistance and SAR.

Plant materials and growth conditions
The sid2 regime in plant growth rooms with 70% humidity.

Mutant screen and characterization
To screen for mutants with SAR defects, wild-type Col-0 seeds were

Plasmid construction and Arabidopsis transformation
For transgenic complementation analysis, a 7.7 kb genomic DNA fragment was amplified by PCR using primers 5'-cggggtaccatatgggtgtgacaggtatg-3' and 5'-cgcggatcccaatcacaagagcaaacagc-3' from genomic DNA of sard3, and cloned into the binary vector pGREEN229 (Hellens et al., 2000) to obtain pGREEN229-camta3.  conidiophores; 2, one leaf infected with more than five conidiophores; 3, two leaves infected, but with no more than five conidiophores on each infected leaf; 4, two leaves infected with more than five conidiophores on each infected leaf; 5, more than two leaves infected with more than five conidiophores. spores at a concentration of 5×10 4 spores/ml two days later. Infection was scored seven days later as described in Figure 1.    Table S1.
The sard3 mutation was flanked between markers F12A24 and F27D4.   Table S1. Mapping markers and their primer sequences. Mb-11 Mb on Chromosome 2. Figure S1. Alignment of the IQ domains of the CAMTA protein family in

Arabidopsis.
The arrow indicates the mutation site in sard3, where Ala is changed to Val.