Iron-sulfur cluster protein NITROGEN FIXATION S-LIKE 1 and its interactor 1 FRATAXIN function in plant immunity 2

30 Iron-sulfur (Fe-S) clusters are inorganic cofactors that are present in all kingdoms of life as part 31 of a large number of proteins involved in several cellular processes, including DNA replication 32 and metabolism. In this work, we demonstrate an additional role for two Fe-S cluster genes in 33 biotic stress responses in plants. Eleven Fe-S cluster genes, including the NITROGEN FIXATION 34 S ( NIFS ) -LIKE 1 ( NFS1 ) and its interactor FRATAXIN ( FH ), when silenced in Nicotiana 35 benthamiana , compromised nonhost resistance to Pseudomonas syringae pv. tomato T1. 36 NbNFS1 expression was induced by pathogens and salicylic acid. Arabidopsis thaliana atnfs and 37 atfh mutants, with reduced AtNFS1 or AtFH gene expression, respectively, showed increased 38 susceptibility to both host and nonhost pathogen infection. Arabidopsis AtNFS1 and AtFH 39 overexpressor lines displayed decreased susceptibility to infection by host pathogen P. syringae 40 pv. tomato DC3000. The AtNFS1 overexpression line exhibited constitutive upregulation of 41 several defense-related genes and enrichment of gene ontology terms related to immunity and 42 salicylic acid responses. Our results demonstrate that NFS1 and its interactor FH are involved 43 not only in nonhost resistance but also in basal resistance, suggesting a new role of the Fe-S 44 cluster pathway in plant immunity. 45 46 47 48 49 50 51 52 53 54


INTRODUCTION
ataxia that leads to premature death (Babcock et al., 1997). In Arabidopsis, atfh knockout 117 mutants were shown to be lethal, but a few AtFH knockdown lines are viable and showed 118 retarded growth, reduced fresh weight and seed number (Busi et al., 2006;Balk and Shaeddler, 119 2014). 120 In plants, the biological role of Fe-S proteins in genotoxic stress and abiotic stress is gradually 121 emerging (Liang et al., 2014;Inigo et al., 2016). There are no reports thus far that directly show 122 Fe-S cluster genes' involvement in plant biotic stress resistance/tolerance. Identification of genes 123 and pathways involved in plant disease resistance is important to develop disease-resistant crops 124 either by breeding or biotechnological approaches. One of the problems of using classical 125 resistance (R) genes in crops to confer disease resistance is their durability because R genes are 126 often effective against a specific pathogen strain. Nonhost resistance (NHR) on the other hand 127 can be used to confer broad and durable disease resistance (Mysore and Ryu, 2004;Gill et al., 128 2015;Lee et al., 2016;Lee et al., 2017;Niks and Marcel, 2009). NHR is a type of resistance 129 shown by all plant species against most potential pathogens (nonhost pathogens) and results 130 usually in compromised or failure to establish virulence (Heath, 2000;Mysore and Ryu, 2004). 131 Several genes involved in NHR have also been used to confer resistance against economically 132 important diseases in crop plants (reviewed in Fonseca and Mysore, 2019). 133 In this study, we demonstrate the role of Fe-S cluster genes in NHR and plant immunity. We 134 identified 11 Fe-S cluster genes to play a role in NHR by virus-induced gene silencing (VIGS) in 135 Nicotiana benthamiana. We further characterized the role of the mitochondrial AtNFS1 and its 136 interactor AtFH in NHR in Arabidopsis using mutants and overexpression lines that exhibited 137 enhanced susceptibility and increased disease resistance, respectively, against host and nonhost 138 pathogens. This is the first report showing the role of Fe-S cluster genes in combating plant 139 biotic stresses.

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Virus-induced gene silencing of the NbNFS1 gene results in increased susceptibility to 144 nonhost bacterial pathogens in N. benthamiana 145 A forward genetics approach using the Tobacco rattle virus (TRV)-based VIGS system to 146 silence genes induced by a mixed cDNA elicitor library (del Pozo et al., 2004;Anand et al., 147 2007; Senthil- Kumar and Mysore, 2014;Senthil-kumar et al., 2018) has been used to 148 successfully identify several genes involved not only in NHR but also in host resistance such as 149 gene-for-gene and pathogen-associated molecular patterns (PAMP)-mediated immunity (Rojas et 150 al., 2012;Senthil-Kumar et al., 2013;Kaundal et al., 2017;Nagaraj et al., 2016). One of the 151 cDNA clones identified in this screening, NbME26D10 (https://vigs.noble.org), displayed 152 homology to the AtNFS1 gene from Arabidopsis. AtNFS1 is annotated to encode a long 153 (AtNFS1-1) and a short (AtNFS1-2) isoform protein, which likely originated due to alternate 154 splicing. The AtNFS1 amino acid sequence is well conserved across plants, animals and even 155 prokaryotes as shown by multiple alignment (Supplemental Figure S1). 156 To further confirm that NFS1 plays a role in NHR, two VIGS constructs were generated with 157 distinct regions of the NbNFS1 coding sequence (NbNFS1-a; 522-822 bp and NbNFS1-b; 120-158 420 bp), for the downregulation of NbNFS1 in N. benthamiana (Supplemental Figure S2A). 159 The TRV2:GFP construct was used as a control (GFP has no sequence similarity to plant 160 genomic DNA and thus will not cause gene silencing) to rule out the possibility of TRV or pathogens, we hypothesized that other Fe-S pathway genes may also play a role in NHR.

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Silencing of Fe-S cluster genes in N. benthamiana compromises nonhost resistance 193 One of the Fe-S genes, NbNFS1, when silenced, resulted in enhanced susceptibility to nonhost 194 pathogens. Therefore, we sought to investigate if other Fe-S cluster pathway genes also play a 195 role in plant defense responses. To do this, we obtained gene sequences for all curated Fe-S 196 cluster genes available in Arabidopsis (Balk and Lobreaux, 2014;Balk and Pilon, 2011). We 197 identified N. benthamiana homologs using a cut-off e-value of 1e-10 for these genes using 198 BLASTX and N. benthamiana Genome v1.0.1 predicted protein database from the Sol Genomics 199 Network (SGN) website (https://solgenomics.net/) (Supplemental Table S1). We then used the  Table S1). 203 Twenty three Fe-S cluster genes were cloned into the TRV2-VIGS binary vector and 204 transformed into A. tumefaciens strain GV2260. VIGS was performed individually for each of 205 the 23 genes in N. benthamiana. Three-weeks later, silenced plants were inoculated with a Interestingly, a gene that encodes a known interactor of NFS1, NbFH (Turowski et al., 2012), 209 when silenced, also compromised NHR against P. syringae pv. tomato T1 at 2 and 3 days after 210 pathogen infection (Fig. 2). Some of the other Fe-S cluster genes that, when silenced, 211 compromised NHR at 1, 2 and 3 days after pathogen infection include two mitochondrial 212 ferredoxin genes that display high sequence identity (87%) between them:  Table S1 and Supplemental Figure S4). These results suggest that Fe-S cluster proteins play a 223 role in plant defense especially for NHR.

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The Fe-S cluster biosynthesis genes AtNFS1 and AtFH contribute to Arabidopsis resistance 225 against host and nonhost pathogens 226 To determine if the role of Fe-S cluster genes in NHR is conserved in other plant species, we 227 obtained Arabidopsis mutants for AtNFS1 and AtFH. We obtained an AtNFS1 heterozygous mutant from the GABI-KAT collection with a T-DNA insertion located on exon 2 (GABI-KAT 229 211c08), referred to here as atnfs1-7, and a homozygous SALK line with an insertion located in 230 the 5' untranslated region (UTR) (SALK_083681C), referred to as atnfs1-2 (Supplemental 231 Figure S5). Since complete loss of NFS1 leads to lethality or severe developmental defects with 232 no viable seed production, we could not obtain a homozygous atnfs1 complete knockout line 233 (Couturier et al., 2013;Balk and Shaeddler, 2014). The atnfs1-7 heterozygous mutant showed 234 reduced AtNFS1 expression (Supplemental Figure S5) and shorter primary root length when were inoculated with a host pathogen P. syringae pv. tomato DC3000 and the nonhost pathogens 238 P. syringae pv. tabaci and P. syringae pv. phaseolicola by flood inoculation (Ishiga et al., 2011, 239 2017), and samples were collected at 0 h and 3 days after infection for bacterial quantification. In 240 agreement with our previous N. benthamiana NbNFS1-silenced plants (Fig. 1), both atnfs1 241 mutants were also susceptible to a nonhost pathogen 3 days after pathogen infection (Fig. 3E   242 and F). Both mutants were also susceptible to the host pathogen P. syringae pv. tomato DC3000 243 3 days after pathogen infection when compared to the WT plants (Fig. 3B, C). The observed 244 difference in Log 10 colony-forming units (CFU) between the atnfs1-7 mutant and the WT (Fig.   245 3B, E), for example, is equivalent to a three-fold-change increase for the atnfs1-7 mutant. The

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Arabidopsis atnfs1-7 mutant also showed disease-related symptoms such as necrosis and wilting 247 three days after infection compared to WT plants (Fig. 3A, D). Similar to NbNFS1 (Fig. 1), the 248 AtNFS1 transcript was also significantly induced by the nonhost pathogens and 1 mM SA in WT

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The susceptibility of two atfh mutants to host and nonhost pathogens was also tested. The significantly reduced compared to the WT control (Supplemental Figure S7). We performed a 257 pathogen flood inoculation assay using both athf1 and atfh2 mutants and WT control plants upon 258 infection with the nonhost pathogen P. syringae pv. phaseolicola and the host pathogen P. 259 syringae pv. tomato DC3000. Similar to NbFH VIGS-silenced plants (Fig. 2), the Arabidopsis 260 atfh1 and atfh2 mutant lines were highly susceptible to nonhost pathogens 3 days after pathogen 261 infection when compared to WT Col-0 ( Fig. 4A, B). In addition, both atfh1 and atfh2 mutants 262 also supported more host bacterial multiplication when compared to WT Col-0 at 3 days after 263 pathogen infection (Fig. 4C). Differences in Log 10 CFU observed between the atfh mutants and 264 the WT is equivalent to a two-fold change increase for the atfh mutant. (Fig. 4D). Similar to the  the AtNFS1 short and long isoforms have a role in disease resistance (Fig. 5A, B). Additionally, 288 the overexpression lines also showed less disease symptoms, such as wilting and water soaked 289 lesion formation, compared to WT plants upon leaf infiltration using a needleless syringe with P. 290 syringae pv. tomato DC3000 (CFU = 8 x 10 6 ) 3 days after infection (Fig. 5C). We also tested the  Figure S12) in a similar manner to the resistance observed against P. 296 syringae pv. tomato DC3000 shown above.

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In addition to AtNFS1 overexpression lines, we also generated two Arabidopsis AtFH 298 overexpression lines . Upon flood inoculation of both FH-OX lines 299 with the host pathogen P. syringae pv. tomato DC3000, similar to AtNFS1-OX lines, both AtFH-300 OX lines showed reduced disease symptoms and accumulated less bacteria when compared to 301 WT Col-0 3 days after pathogen infection (Fig. 5D). We did not observe any obvious phenotypic    Nelson et al., 2007). Two days after the co-infiltration, leaves were observed under a confocal 346 microscope and co-localization of AtNFS1:GFP and mCherry could be observed specifically in 347 the mitochondria but not in the chloroplast (Supplemental Figure S14). 349 To understand the mechanism of reduced susceptibility to host pathogens in AtNFS1  Table S3). Subsequently, these 85 upregulated DEGs were functionally classified according to 362 gene ontology (GO) terms using AgriGO2 (Tian et al., 2017) (Supplemental Table S4). To  Table S4). Interestingly, 12 overrepresented GO terms belong to 367 defense-related processes such as immune response, innate immune response, response to biotic 368 stimulus, systemic acquired resistance and SA-mediated signaling pathway. We also found 29 369 GO terms overrepresented (FDR < 0.05) for the same dataset of 85 upregulated DEGs using  Table S5). Similar to AgriGO2, the same 371 defense-related GO pathways were found to be enriched using Panther such as response to SA, 372 systemic acquired resistance and immune responses (Fig. 8B). In addition to defense-related 373 processes, we also observed overrepresentation of GO terms related to Fe-S clusters such as 374 metal ion binding, cofactor binding, and adenyl nucleotide binding (Supplemental Table S4; 375 Supplemental Figure S15B). Additionally, using AgriGO2 and Panther, we also identified 376 overrepresented GO terms from other biological processes that may also play a role in plant 377 defense such as response to oxygen and response to stress (Supplemental Tables S4 and S5). 378 Strikingly, a total of 53 upregulated DEGs were identified at the 0 h basal control (fold change 379 >1; FDR < 0.05) in the NFS1.2-18-OX line compared to the WT (Supplemental Table S6).  Table S7) but no molecular functions 384 related to defense responses were found, and other GO terms like hydrolase activity and binding 385 were identified from these DEGs. 386 We validated the differential expression of seven upregulated defense-related genes (0 h time   Furthermore, we showed that the nuclear-encoded AtNFS1:GFP co-localized in mitochondria 413 along with the mitochondria tracker mCherry. This rules out a possible plastidial localization for 414 AtNFS1 and further confirms previous studies (Frazzon et al., 2007).

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In addition to NbFS1, we show that 11 out of 23 other Fe-S cluster genes tested also play a role 416 in NHR in N. benthamiana. Along with NFS1, we also further characterized its interactor, FH was shown that GRX480 transcripts were significantly induced by SA (Ndamukong et al., 2007). 433 We also investigated the role of the AtNFS1 interactor protein, AtFH, in disease resistance.

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In conclusion, the genetic and transcriptomic data, in combination with disease phenotype assays 482 presented here suggest that the Fe-S cluster interactor proteins NFS1 and FH are involved not 483 only in NHR but also in basal resistance. We propose a new role for Fe-S cluster genes in plant 484 immunity responses that has been overlooked until now. Our results also suggest that NFS1, its interactor FH, and possibly other Fe-S cluster genes could be new targets for crop breeding and 486 genetic engineering to generate durable disease-resistant varieties.  Table S9).  Invitrogen) and then transformed into Agrobacterium tumefaciens strain GV3101 for stable 515 transformation in Arabidopsis using the floral dip method (Clough and Bent, 1998). All cloned 516 genes were checked by sequencing using Sanger sequencing. The 907 bp promoter region of 517 AtNFS1 was amplified by PCR from genomic DNA (50 ng) and cloned into the GUS reporter 518 binary vector pMDC162 by GATEWAY cloning and stably-transformed into Arabidopsis in the 519 same way as described above for GUS histochemical studies. All primers used in this work can 520 be found in Supplemental Table S1.  containing the following nonhost pathogens: P. syringae pv. tomato T1 and P. syringae pv. 559 glycinea at 8x10 5 CFU. We collected tissues at 0, 1, 2 and 3 days after infection. Two leaf-disks 560 of 10 mm diameter were collected per plant from at least 5 plants (n = 5) using a 0.5 cm 2 borer.

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Leaf discs were placed in tubes containing grinding beads and 500 µL of sterile distilled water 562 and ground to obtain a homogeneous suspension. Suspensions of 20 µL were transferred to a 563 plate containing 180 µL of sterile distilled water and 10× serial dilutions were made. Aliquots of 564 10 µL were then plated on KB medium with the appropriately selected antibiotic and incubated 565 at 30°C for two days, after which CFU were counted. We monitored bacterial growth using 566 GFPuv-labeled P. syringae strains two to three days after infection in N. benthamiana. Statistical 567 analyses were done using Student's t test of the differences between two means of log-568 transformed data or by one-way ANOVA using the GraphPad Prism software.  30 seconds using LI-8x0 v1.02 software. Specific shoot respiration rate (nmol CO 2 gfw -1 s -1 ) was 654 calculated from the raw CO 2 flux data using R version 3.6.0 where a linear regression was used 655 to calculate the CO 2 flux rate with dead band set at 60 seconds, and then divided by total shoot 656 fresh weight.     CFU/mL. Samples (rosettes) were collected at 0 h and 3 dpi for bacterial quantification.

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All experiments were repeated two times with similar results. week-old WT, atfh1 and atfh2 plants were flood inoculated with P. syringae pv. phaseolicola at 8 817 x 10 5 CFU/mL. Samples (rosettes) were collected at 0 h and 3 dpi for bacterial quantification.

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Histograms represent means of four biological replicates. Error bars indicate standard error.