Arabidopsis plants were grown in soil at 23°C/21 °C day/night under 16/8-h light/dark cycles in a growth chamber with 40% relative humidity (RH) (Bi et al., 2010). The N. benthamiana plants were sowed and grown in a controlled environment room (CER) at 22 °C and 45–65% humidity with a 16/8-h light/dark cycles (Lin et al., 2023). Four-week-old plants were used for assay. The potato plants were grown in an artificial climate chamber at 25 ± 2 °C and 58–67% relative humidity under a 16/8-h light/dark photoperiod (Yang et al., 2023).

Mapping‐by‐sequencing involves combining next-generation sequencing with classical genetic mapping to identify candidate mutations associated with a phenotype was carried out as previously described (Sun et al., 2020). The mutant phenotype of the selected F₂ lines were confirmed by examining the self‐fertilized F₃ progeny. Leaves were collected from the F₃ progeny of 30 F₂ lines with confirmed mutant-like phenotype. Genomic DNA was extracted from the mixed tissue and sent for WGS. WGS reads were aligned with the TAIR10 reference genome. SNPs were identified and the ratios of SNPs were plotted and used for linkage analysis. Genes containing nonsynonymous mutations in the linkage region were selected as candidate genes for knockout analysis.

The laz1 mutations were generated by targeting AT4G38360 in sard1-1 and Col using the egg cell- specific promoter-controlled CRISPR/Cas9 system (Wang et al., 2015). The laz1h1 mutations were generated by targeting AT1G77220 in sard1-1 and Col, respectively, using the same CRISPR/Cas9 system. The sard1-1 sard6-2 F₂ mutant was obtained by crossing sard1-1 with SALK_023954C (laz1-7). The sard1-1mutant was reported (Zhang et al., 2010). Refer to Supplementary Table S1 for a comprehensive list of all primers utilized in this process.

Total RNA was extracted from various tissues using TRIzol reagent (Invitrogen). Complementary DNAs (cDNAs) were synthesized using a ReverTra Ace kit (Toyobo) and served as templates for quantitative reverse transcription polymerase chain reaction (qRT-PCR), which was conducted with a SYBR Premix ExTaq kit (Takara) on a Bio-Rad iQ2 system. The procedure was as follows: initial polymerase activation for 30 s at 95°C followed by 40 cycles of 95°C for 5 s and 60°C for 20 s (Chen Y. et al., 2021). Each sample underwent three biological replicates and three technical replicates. The expression levels of the target genes were normalized to those of the actin gene. Primers used for qPCR can be found in Supplementary Table S1 .

The obligate pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2 spore suspension in water was weekly propagated on Col seedlings at 18 °C and 60-80% humidity with a 12/12-h light/dark cycles (Zhang et al., 2010; Bi et al., 2010). The Phytophthora infestans (P. infestans) strain 1306 were cultured on Rye A agar medium at 18°C in the dark. The Phytophthora capsici (P. capsici) strain BYA5 was cultured on Rye A agar medium at 24°C in the dark (Abrahamian et al., 2016; Wang et al., 2019). The bacterial pathogen Pseudomonas syringae pv maculicola (Psm) ES4326 was cultured on King’s B medium Agar plate supplemented with 50 μg/mL streptomycin at 28°C incubator (Zhang et al., 2010).

For infection with Psm ES4326 (diluted in 10 mM MgCl₂ to OD₆₀₀ as indicated below) or 10 mM MgCl₂, leaves of 3-week-old plants were infiltrated with the bacteria at a dose of OD₆₀₀ = 0.0025-0.005 for SAR and OD₆₀₀ = 0.001 for gene expression. For SAR assay (Zhang et al., 2010), the Hpa Noco2 infection assay was carried out on 3-week-old soil-grown seedlings two days after infection with Psm ES4326, by spraying plants with Hpa Noco2 spore suspension at a concentration of 5 x 10⁴ spores/mL. Inoculated plants were covered with a clean dome and grown at 18 °C under 12/12-h light/dark cycles in a growth chamber and growth of Hpa Noco2 was quantified seven days later. For genes expression, infected leaves were collected at two days after inoculation, two or three infected leaves of different plants were collected as one sample, and three samples were used for each genotype (Bi et al., 2010; Lan et al., 2023).

For inoculation assay with P. infestans strain 1306 (Abrahamian et al., 2016) and P. capsici strain BYA5 (Wang et al., 2019) on Nb leaves, Agrobacterium tumefaciens cultures were resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES [pH 5.6], and 150 μM acetosyringone) at a final concentration of OD₆₀₀ = 0.6 and infiltrated into leaves for transient expression of interested genes in planta. The leaves were detached 48h after agroinfiltration, then inoculated with P. capsici BYA5 mycelium (r = 2.5 mm) or 10 μL of the P. infestans 1306 zoospore suspension (200 zoospores/μL). The lesion areas (cm²) of P. capsici-inoculated leaves were measured under UV light at 48 h after inoculation. The P. infestans-inoculated leaves were incubated in a growth chamber at 18°C, and lesion areas were scored 3-4 days after infection.

The total SA was extracted following a modified method previously described for extraction of phenolic compounds (Zhang et al., 2012). About 100 mg of leaf tissue from 3- or 4-week-old plants 2 days after inoculation with Psm ES4326 (OD₆₀₀ = 0.001) was collected, in four biological replicates from independent plants for each genotype. The rosette leaves were ground in liquid nitrogen. Around 100mg powders were added into 1ml 80% MeOH in a 2ml eppendorf tube. Then the eppendorf tube was agitated for 2hr at 4°C, and then centrifuged at 13,000g at 4°C for 10 min. The supernatant was transferred into a new eppendorf tube, and the sediment was re-extracted with 500μl 100% MeOH. Both extracts were combined and blow-dryed by nitrogen gas, then was resolved by 500μl sodium acetate (0.1M, pH 5.5). The resuspension was added with 10 μl β-glucosidase (1Uμl-1) and hydrolyzed at 37°C for 2hr in the water bath. After the hydrolysate was heated in boiling water for 5 min and centrifuged at 13,000g at 4°C for 10 min, the supernatant was used for analyzing total SA by HPLC as mentioned previously (Zhang et al., 2012). SA was detected at 296-nm excitation and 410-nm emission by using fluorescence detector. According to the standard curve, the concentration of SA is calculated by the HPLC peak area.

As shown in Figures 1A, B , wild-type (Col) plants were susceptible to the virulent isolate of Hyaloperonospora arabidopsidis (Hpa) Noco2. After treatment with the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) ES4326, Col plants became resistant against Hpa Noco2, suggesting an robust SAR response induced by Psm infection. The sard1-1 plants showed a mild SAR-compromised phenotype, while the sard1-1 sard6-1 double mutant exhibited an exacerbated SAR-deficient phenotype. We examined Psm-induced expression of the defense marker genes PR1 and PR2 in those lines and found that induction of both genes in sard1-1 sard6-1 double mutant by Psm treatment was significantly reduced compared to that in sard1-1 mutant ( Figures 1C, D ). We also detected a further compromised induction of critical genes involved in SA biosynthesis CBP60g and ICS1 in the sard1-1 sard6-1 mutant compared to sard1-1 mutant ( Figures 1E, F ). Next, we quantified SA levels in Col, sard1-1 and the sard1-1 sard6-1 mutant plants. Following treatment with Psm, the total SA levels in the sard1-1 sard6-1 mutant was significantly reduced compared to those in the Col or sard1-1 plants ( Figure 1G ). These findings indicate that the systemic resistance in the sard1-1 sard6-1 mutant may be impeded due to the impact on the induction of CBP60g and SA biosynthesis.

To identify sard6, we performed combining next-generation sequencing with classical genetic mapping to identify candidate mutations associated with a phenotype on sard1-1 sard6-1 mutant. The sard1-1 sard6-1 mutant was backcrossed with the sard1-1 line and resulting F₁ plants exhibit sard1-like sard phenotype ( Figure 2A ; Supplementary Figure S1A ), suggesting sard6-1 is a recessive mutant. In the F₂ generation, lines with sard1-1 sard6-1-like and sard1-like sard phenotype were kept and validated in the F3 progeny, respectively. Pooled genomic DNA from each segregant population (30 confirmed lines) was subjected to whole-genome next-generation sequencing (WGS). Analysis of the single nucleotide polymorphism (SNP) frequency distribution across the genome unveiled a linked genetic region on chromosome 4 ( Supplementary Figure S1B ). Within this chromosomal segment, three genes, AT4G30790, AT4G30990 and AT4G38360, using linkage analysis, we detected G to A transitions that resulted in missense mutations ( Figure 2B ). To ascertain the gene correlating with the sard1-1 sard6-1 phenotype, we generated deletion mutant for each gene in sard1-1 background using CRISPR/Cas9 technology ( Figure 2C ). Upon subsequent SAR analysis of the homozygous lines for the three candidate genes, only sard1-1 AT4G38360-cr double mutant phenocopied sard1-1 sard6-1, indicating that AT4G38360, alias LAZARUS1 (LAZ1) (Malinovsky et al., 2010), is the gene of interest ( Figure 2D ).

To further ascertain the association of the mutation in AT4G38360 with the sard1-1 sard6-1 phenotype, we identified the sard1-1 sard6-2 double mutant from F₂ progeny of a cross between sard1-1 and sard6-2, a T-DNA mutant SALK_023954C targeting the At4G38360 locus and silencing the gene ( Supplementary Figures S2A, S2C ), and confirmed DNA fragment deletion in sard1-1 AT4G38360-cr double mutant (reassigned as sard1-1 sard6-3) using Sanger sequencing ( Supplementary Figure S2B ). Subsequent SAR verification revealed that both sard1-1 sard6-2 and the sard1-1 sard6-3 lines exhibited sard1-1 sard6-1-like sard phenotype ( Figure 2E ). Additionally, we performed genetic complementation by agrobacteria mediated transformation of a 4.3 kb fragment containing LAZ1 coding region into the sard1-1 sard6-1 mutant. Three independent lines with LAZ1 expression were chosen and tested for SAR phenotype ( Supplementary Figure S2D ). As shown in Figure 2F , these three lines exhibited sard1-like sard phenotype, indicating that expressing LAZ1 revert the enhanced sard phenotype of sard1-1 sard6-1. These findings support that the SARD6 locus corresponds to AT4G38360/LAZ1, encoding a protein with a DUF300 domain. This protein is implicated in vacuolar transport and appears to modulate brassinosteroid signaling pathways (Malinovsky et al., 2010). For simplicity and consistency, sard6-1, sard6-2 and sard6-3 is reassigned to laz1-6, laz1-7 and laz1-8, respectively ( Supplementary Figure S2A ).

In Arabidopsis, LAZ1 has a close homolog, LAZ1 Homolog 1 (LAZ1H1, AT1G77220). Quantitative PCR analysis of the expression of LAZ1 and LAZ1H1 showed that both genes were induced after Psm ES4326 treatment ( Supplementary Figures S3A, B ), indicating that, like LAZ1, LAZ1H1 may also play a role in plant immunity. To check whether LAZ1H1 contributes to SAR, we employed CRISPR/Cas9 technology to generate knockout mutants for LAZ1H1 in both the Col and sard1-1 backgrounds. Utilizing PCR amplification and sanger sequencing, we identified three homozygous deletion lines in the sard1-1 background and three deletion lines in Col, designated as sard1-1 laz1h1-1, sard1-1 laz1h1-2, sard1-1 laz1h1-3, laz1h1-4, laz1h1-5 and laz1h1-6, respectively ( Supplementary Figure S3C ). Upon verification of SAR response in the sard1-1 laz1h1 lines, we observed that they exhibited the same phenotype as the sard1-1 laz1-8 double knockout, while laz1h1 mutants showed minimal sard phenotype ( Supplementary Figure S3D ), suggesting that LAZ1H1 also positively regulates SAR.

To investigate the roles of LAZ1 and LAZ1H1 in SAR, we employed CRISPR/Cas9 technology to generate knockout mutants for laz1 in Col background. Utilizing PCR amplification and sanger sequencing, we identified two homozygous deletion lines in Col, designated as laz1-9, laz1-10 respectively ( Supplementary Figure S2B ), and generated laz1-7 laz1h1-5 double mutant from F₃ progeny of a cross between laz1-7 and laz1h1-5. Psm-induced SAR in Col, laz1-7, laz1-9, laz1-10, laz1h1-5, laz1-7 laz1h1-5 plants were performed. Compared to Col, the mutants laz1-7, laz1-9 and laz1-10, laz1h1-5 exhibited a weak sard phenotype, laz1-7 laz1h1-5 exhibited a stronger sard phenotype ( Figure 3A ).

We examined Psm-induced expression of SARD1 and CBP60g as well as biosynthetic genes of SA and NHP in Col, laz1-7, laz1h1-5 and laz1-7 laz1h1-5 lines and found that induction of these genes in three mutants were significantly reduced compared to that in Col, and the expression levels of ICS1, EDS5, PBS3, ALD1, SARD4 and FMO1 were lower in laz1-7 laz1h1-5 ( Figures 3B–D ). The results showed that laz1 and laz1h1 play overlapping roles in plant immunity.

Given that NHP acts as the mobile signal for SAR (Chen et al., 2018; Hartmann et al., 2018) and that the LAZ1/LAZ1H1 are putative channel proteins, we investigated whether LAZ1/LAZ1H1 is necessary for NHP-induced immune responses. Col, along with the laz1-7, laz1-9, laz1-10, laz1h1-4, laz1h1-5, laz1h1-6 and laz1-7 laz1h1-5 mutants were utilized as experimental materials for verification. Initially, we infiltrated the primary leaves with 1 mM or 0.3 mM NHP and subsequently spray-inoculated the entire plants with a spore suspension of Hpa Noco2, separately. As depicted in Supplementary Figure S4 , minimal pathogen growth was observed on Col pretreated with NHP, indicating that NHP confers robust resistance against Hpa Noco2. Similar outcomes were observed in the NHP-pretreated seven mutant lines. These results suggest that laz1 and laz1h1 are not involved in the regulation of NHP-induced immunity.

The results mentioned above suggest that LAZ1 and LAZ1H1 positively regulate immunity against the obligate oomycete Hpa Noco2. To determine if LAZ1 and LAZ1H1 could augment resistance against different Phytophthora pathogens, we individually inserted the genomic sequences of LAZ1 and LAZ1H1, including 35S promoters, coding regions, and terminators, into the pCAMBIA1300 vector. These constructs were transformed into agrobacterium and used for agrobacterium-mediated transient overexpression of the respective proteins in Nicotiana benthamiana (Nb) leaves. After 48 hours post-infiltration of agrobacterium strain carrying 35S-LAZ1, 35S-LAZ1H1 or empty vector, the Nb leaves were inoculated with spores of P.infestans strain 1306 or mycelium of P.capsici strain BYA5. The resulting lesion areas were evaluated 3-4 days following infection. In Figure 4A , Nb leaf areas overexpressing LAZ1 exhibited significantly reduced lesion sizes compared to those with an empty-vector (EV) control after inoculation with P.infestans strain 1306, with statistically significant differences in lesion sizes observed ( Figure 4B ). Overexpression of LAZ1H1 yielded analogous results ( Figures 4C, D ).

Similarly, overexpression of LAZ1 or LAZ1H1 lead to reduced lesion sizes compared to the EV control, following infection with P.capsici strain BYA5 ( Figures 4E–H ). These findings indicate that overexpression of LAZ1 or LAZ1H1 in Nb leaves can enhance resistance to P.infestans and P.capsici.

Homologs of LAZ1 were found in various plants. To understand whether homologs of the LAZ1 gene in N.benthamiana and Solanum tuberosum (potato) can enhance resistance to Phytophthora, we identified sequences with high homology to LAZ1 from the N.benthamiana genome database (https://nbenthamiana.jp/nbrowser/anno) (Kurotani et al., 2023) and the genome database of the diploid potato inbreeding line A157 (Zhang et al., 2021). The protein sequences of Nbe.v1.s00130g02480 (NB00130g02480) and Nbe.v1.s00150g07560 (NB00150g07560) in N.benthamiana and the protein sequences of A157_07G018790 and A157_12G022720 in A157 showed the highest homology to LAZ1 protein ( Figure 5A ). Phylogenetic analysis grouped these four genes into a single cluster, suggesting a close evolutionary relationship, and LAZ1 is closely related to these four proteins ( Supplementary Figure S5A ). The coding sequences (CDS) of these four homologs gene were amplified and individually inserted into the binary pCAMBIA1300 vector under the 35S promoter. Transient expression in Nb leaves followed by inoculation with P.infestans strain 1306 revealed that the lesion areas at sites of overexpression of these four genes were significantly reduced compared to the empty-vector (EV) control ( Figure 5B ), with statistically significant differences observed ( Figure 5C ). Validation through western blot analysis confirmed the presence of protein products encoded by these genes in Nb leaves ( Supplementary Figure S5B ). These findings indicate that overexpression of these LAZ1 homologs significantly enhances the resistance of N. benthamiana to P.infestans.

LAZ1 and LAZ1H1 belong to the evolutionarily conserved DUF300 family of transmembrane proteins in eukaryotes. LAZ1 serves as a regulatory factor for certain Hypersensitive Response (HR) cell deaths conditioned by the TIR-NB-LRR protein RPS4 and by the CC-NB-LRR protein RPM1 (Malinovsky et al., 2010). LAZ1 and LAZ1H1 have been shown to play a pivotal role in maintaining vacuole membrane integrity, which is crucial for proper Brassinosteroid (BR) signaling (Liu et al., 2018). Despite these insights, the roles of LAZ1 and LAZ1H1 in the systemic acquired resistance (SAR) pathway remain unexplored. In this report, we show that LAZ1 and LAZ1H1 are positive regulators of SAR.

Employing a forward genetic strategy, we isolated the sard1-1 sard6-1 mutant as one of sard enhancers of sard1-1. This sard1-1 sard6-1 mutant exhibited severely impaired systemic resistance ( Figures 1A, B ). Through mapping-by-sequence and gene complementation, we identified that SARD6 encodes LAZ1 ( Figure 2 ). We also found that loss-of-function of LAZ1H1 leads to enhanced SAR deficiency in sard1-1 background ( Supplementary Figure S3 ). Furthermore, we showed that the laz1 laz1h1 double mutant exhibited stronger sard phenotype compared to laz1 and laz1h1 single mutants ( Figure 3A ), indicating a functional redundancy between LAZ1 and LAZ1H1 in regulation of SAR.

In this study, we have used Psm ES4326, a virulent pathogen which does not trigger HR, to induce SAR and found that Laz1 and laz1h1 mutants are compromised in SAR, suggesting that LAZ1/LAZ1H1 play an HR-independent role in SAR.

SA and NHP are two key signaling molecules in SAR and expression levels of their biosynthetic genes are tightly controlled during plant immunity (Hartmann and Zeier, 2019; Peng et al., 2021). SARD1 and CBP60g are master transcriptional regulators in plant defense and positively regulate biosynthetic genes of SA and NHP (Sun et al., 2020). Psm-induced expression of CBP60g and ICS1 in sard1-1 sard6-1 was further diminished compared to that in sard1-1 ( Figures 1E, F ) and total SA levels was lower in sard1-1 sard6-1 than that in sard1-1 ( Figure 1G ), indicating that the involvement of LAZ1 in SAR may be attributed to its regulatory effects on the expression of CBP60g and ICS1, as well as the accumulation of SA.

laz1 and laz1h1 single mutants showed minor sard phenotype while laz1 laz1h1 double mutant exhibited stronger sard phenotype ( Figure 3A ). Accordingly, Psm-induced expression of biosynthesis genes of SA and NHP in laz1 and laz1h1 single mutants was reduced compared to that in Col, and further diminished in laz1 laz1h1 double mutant ( Figures 3C, D ), suggesting that LAZ1 and LAZ1H1 have an overlapping function in SAR and that they positively regulate SAR by modulating the expression of biosynthetic genes of SA and NHP. Since Psm-induced expression of SARD1 and CBP60g was diminished in laz1 laz1h1 double mutant ( Figure 3B ), it is possible that LAZ1 and LAZ1H1 modulate the expression of biosynthetic genes of SA and NHP through regulating expression of SARD1 and CBP60g. In addition, we showed that NHP-induced resistance against Hpa Noco2 was not significantly affected in laz1, laz1h1 or laz1 laz1h1 double mutant ( Supplementary Figure S4 ). These results suggest that LAZ1 and LAZ1H1 regulate SAR mainly through affecting the expression of biosynthesis genes of SA and NHP. However, the underlying mechanism necessitates further investigation.

Recently, LAZ1 homologs in maize, ZmLAZ1-4 and ZmLAZ1-8, were predicted to bind metal ions including Zn²⁺, Mg²⁺, or Ca²⁺ and ZmLAZ1-4 protein was shown to act as a Zinc transporter that modulate Zinc homeostasis on plasma and vacuolar membrane (Liu et al., 2022), it will be interesting to test whether LAZ1 and LAZ1H1 combine Ca²⁺ and regulate the expression of defense genes through modulating calcium homeostasis during plant immunity.

LAZ1 and LAZ1H1 are conserved proteins with their homologs found in various plants. We showed overexpression of LAZ1 and LAZ1H1 as well as their close homologs from N.benthamiana and potato in Nb leaves leads to enhanced resistance to Phytophthora species ( Figures 4 , 5 ), suggesting that LAZ1 and LAZ1H1 are evolutionarily conserved in positive regulation of plant defense, thereby emphasizing the significance and utility of these genes in investigating plant-pathogen interactions. Presently, in potato, the cultivation of disease-resistant varieties is mainly to isolate disease resistance genes from wild species and introduce them into cultivated varieties through genetic transformation. The homologs of LAZ1 in potato were found to similarly bolster the plants’ resistance to Phytophthora ( Figures 5B, C ). This study provides an important genetic resource for potato disease resistance breeding.