Engineering Pathogen Resistance in Crop Plants

As the world population continues to increase, food supplies must also grow to meet nutritional requirements. One means of ensuring the stability and plentitude of the food supply is to mitigate crop loss caused by plant pathogens. Strategies for combating disease include traditional technologies such as plant breeding and chemical applications; current technologies such as generating transgenic plants that express components of known defense signaling pathways; and the adaptation of newer technologies such as RNA silencing of pathogen and plant transcripts. Breeding has been used to pyramid resistance (R) genes into many different plants including rice. Chemical strategies include application of salicylic acid (SA) analogs to stimulate systemic acquired resistance (SAR) responses. Genetic screens in Arabidopsis have identified genes controlling SAR and these genes have been manipulated and used to engineer crop plants. The diseases caused by plant viruses are being thwarted through the initiation of endogenous RNA silencing mechanisms. Many of these strategies show great promise, some limitations, and exciting opportunities to develop many new tools for combating plant pests.

BackgroundThe sulfanilamide family comprises a clinically important group of antimicrobial compounds which also display bioactivity in plants. While there is evidence that sulfanilamides inhibit folate biosynthesis in both bacteria and plants, the complete network of plant responses to these compounds remains to be characterized. As such, we initiated two forward genetic screens in Arabidopsis in order to identify mutants that exhibit altered sensitivity to sulfanilamide compounds. These screens were based on the growth phenotype of seedlings germinated in the presence of the compound sulfamethoxazole (Smex).ResultsWe identified a mutant with reduced sensitivity to Smex, and subsequent mapping indicated that a gene encoding 5-oxoprolinase was responsible for this phenotype. A mutation causing enhanced sensitivity to Smex was mapped to a gene lacking any functional annotation.ConclusionsThe genes identified through our forward genetic screens represent novel mediators of Arabidopsis responses to sulfanilamides and suggest that these responses extend beyond the perturbation of folate biosynthesis.

Exine, the outer plant pollen wall, has elaborate species-specific patterns, provides a protective barrier for male gametophytes, and serves as a mediator of strong and species-specific pollen-stigma adhesion. Exine is made of sporopollenin, a material remarkable for its strength, elasticity, and chemical durability. The chemical nature of sporopollenin, as well as the developmental mechanisms that govern its assembly into diverse patterns in different species, are poorly understood. Here, we describe a simple yet effective genetic screen in Arabidopsis (Arabidopsis thaliana) that was undertaken to advance our understanding of sporopollenin synthesis and exine assembly. This screen led to the recovery of mutants with a variety of defects in exine structure, including multiple mutants with novel phenotypes. Fifty-six mutants were selected for further characterization and are reported here. In 14 cases, we have mapped defects to specific genes, including four with previously demonstrated or suggested roles in exine development (MALE STERILITY2, CYP703A2, ANTHER-SPECIFIC PROTEIN6, TETRAKETIDE α-PYRONE REDUCTASE/DIHYDROFLAVONOL-4-REDUCTASE-LIKE1), and a number of genes that have not been implicated in exine production prior to this screen (among them, fatty acid ω-hydroxylase CYP704B1, putative glycosyl transferases At1g27600 and At1g33430, 4-coumarate-coenzyme A ligase 4CL3, polygalacturonase QUARTET3, novel gene At5g58100, and nucleotide-sugar transporter At5g65000). Our study illustrates that morphological screens of pollen can be extremely fruitful in identifying previously unknown exine genes and lays the foundation for biochemical, developmental, and evolutionary studies of exine production.

One recently identified mechanism that regulates mRNA abundance is RNA silencing, and pioneering work in Arabidopsis thaliana and other genetic model organisms helped define this process. RNA silencing pathways are triggered by either self-complementary fold-back structures or the production of double-stranded RNA (dsRNA) that gives rise to small RNAs (smRNAs) known as microRNAs (miRNAs) or small-interfering RNAs (siRNAs). These smRNAs direct sequence-specific regulation of various gene transcripts, repetitive sequences, viruses, and mobile elements via RNA cleavage, translational inhibition, or transcriptional silencing through DNA methylation and heterochromatin formation. Early genetic screens in Arabidopsis were instrumental in uncovering numerous proteins required for these important regulatory pathways. Among the factors identified by these studies were RNA-dependent RNA polymerases (RDRs), which are proteins that synthesize siRNA-producing dsRNA molecules using a single-stranded RNA (ssRNA) molecule as a template. Recently, a growing body of evidence has implicated RDR-dependent RNA silencing in many different aspects of plant biology ranging from reproductive development to pathogen resistance. Here, we focus on the specific functions of the six Arabidopsis RDRs in RNA silencing, their ssRNA substrates and resulting RDR-dependent smRNAs, and the numerous biological functions of these proteins in plant development and stress responses.

Genetic screens are powerful tools to dissect complex biological processes, but a rate-limiting step is often the cloning of targeted genes. Here, we present a strategy, "mutagenomics," to identify causal mutations from a screen in a high throughput fashion in the absence of backcrossing. Mutagenomics is initiated by sequencing the genomes of the mutants identified, which are then subjected to a three-stage pipeline. The first stage identifies sequence changes in genes previously linked to the targeted pathway. The second stage uses heuristics derived from a simulation strategy to identify genes that are represented by multiple independent alleles more often than expected by chance. The third stage identifies candidate genes for the remaining lines by sequencing multiple lines of common descent. Our simulations indicate that sequencing as few as three to four sibling lines generally results in fewer than five candidate genes. We applied mutagenomics to a screen for Arabidopsis (Arabidopsis thaliana) mutants involved in the response to the phytohormone cytokinin. Mutagenomics identified likely causative genes for many of the mutant lines analyzed from this screen, including 13 alleles of the gene encoding the ARABIDOPSIS HIS KINASE4 cytokinin receptor. The screen also identified 1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASE7, an ACC synthase homolog involved in ethylene biosynthesis, and ELONGATED HYPOCOTYL5 (HY5), a master transcriptional regulator of photomorphogenesis. HY5 was found to mediate a subset of the transcriptional response to cytokinin. Mutagenomics has the potential to accelerate the pace and utility of genetic screens in Arabidopsis.

Nucleotide-binding leucine-rich repeat proteins (NLRs) serve as intracellular immune receptors in animals and plants. Sensor NLRs perceive pathogen-derived effector molecules and trigger robust host defense. Recent studies revealed the role of three coiled-coil-type NLRs (CNLs) of the ADR1 family - ADR1, ADR1-L1 and ADR1-L2 - as redundant helper NLRs, whose function is required for defense mediated by multiple sensor NLRs. From a mutant snc1-enhancing (MUSE) forward genetic screen in Arabidopsis targeted to identify negative regulators of snc1 that encodes a TIR-type NLR (TNL), we isolated two alleles of muse15, both carrying mutations in ADR1-L1. Interestingly, loss of ADR1-L1 also enhances immunity-related phenotypes in other autoimmune mutants including cpr1, bal and lsd1. This immunity-enhancing effect is not mediated by increased SNC1 protein stability, nor is it fully dependent on the accumulation of the defense hormone salicylic acid (SA). Transcriptional analysis revealed an upregulation of ADR1 and ADR1-L2 in the adr1-L1 background, which may overcompensate the loss of ADR1-L1, resulting in enhanced immunity. Interestingly, autoimmunity of snc1 and chs2, which encode typical TNLs, is fully suppressed by the adr1 triple mutant, suggesting that the ADRs are required for TNL downstream signaling. This study extends our knowledge on the interplay among ADRs and reveals their complexity in defense regulation.

Xuemei Chen

Small interfering RNAs (siRNAs) are processed from virus-specific dsRNA to direct antiviral RNA interference (RNAi) in diverse eukaryotic hosts. We have recently performed a sensitized genetic screen in Arabidopsis (Arabidopsis thaliana) and identified two related phospholipid flippases required for antiviral RNAi and the amplification of virus-derived siRNAs by plant RNA-dependent RNA polymerase1 (RDR1) and RDR6. Here we report the identification and cloning of ANTIVIRAL RNAI-DEFECTIVE2 (AVI2) from the same genetic screen. AVI2 encodes a multispan transmembrane protein broadly conserved in plants and animals with two homologous human proteins known as magnesium transporters. We show that avi2 mutant plants display no developmental defects and develop severe disease symptoms after infection with a mutant Cucumber mosaic virus (CMV) defective in RNAi suppression. AVI2 is induced by CMV infection, particularly in veins, and is required for antiviral RNAi and RDR6-dependent biogenesis of viral siRNAs. AVI2 is also necessary for Dicer-like2-mediated amplification of 22-nucleotide viral siRNAs induced in dcl4 mutant plants by infection, but dispensable for RDR6-dependent biogenesis of endogenous transacting siRNAs. Further genetic studies illustrate that AVI2 plays a partially redundant role with AVI2H, the most closely related member in the AVI2 gene family, in RDR1-dependent biogenesis of viral siRNAs and the endogenous virus-activated siRNAs (vasi-RNAs). Interestingly, we discovered a specific genetic interaction of AVI2 with AVI1 flippase that is critical for plant development. We propose that AVI1 and AVI2 participate in the virus-induced formation of the RDR1/RDR6-specific, membrane-bound RNA synthesis compartment, essential for the biogenesis of highly abundant viral siRNAs and vasi-RNAs.

Cryptic splice sites in eukaryotic genome are generally dormant unless activated by mutation of authentic splice sites or related splicing factors. How cryptic splice sites are used remains unclear in plants. Here, we identified two cryptic splicing regulators, RBP45d and PRP39a that are homologs of yeast U1 auxiliary protein Nam8 and Prp39, respectively, via genetic screening for suppressors of the virescent sot5 mutant, which results from a point mutation at the 5' splice site (5' ss) of SOT5 intron 7. Loss-of-function mutations in RBP45d and PRP39a significantly increase the level of a cryptically spliced variant that encodes a mutated but functional sot5 protein, rescuing sot5 to the WT phenotype. We furtherly demonstrated that RBP45d and PRP39a interact with each other and also with the U1C, a core subunit of U1 snRNP. We found that RBP45d directly binds to the uridine (U)-rich RNA sequence downstream the 5' ss of SOT5 intron 7. However, other RBP45/47 members do not function redundantly with RBP45d, at least in regulation of cryptic splicing. Taken together, RBP45d promotes U1 snRNP to recognize the specific 5' ss via binding to intronic U-rich elements in plants.

DNA methylation is a conserved epigenetic modification that is typically associated with silencing of transposable elements and promoter methylated genes. However, some DNA-methylated loci are protected from silencing, allowing transcriptional flexibility in response to environmental and developmental cues. Through a genetic screen in Arabidopsis (Arabidopsis thaliana), we uncovered an antagonistic relationship between the MICRORCHIDIA (MORC) protein and the IMITATION SWITCH (ISWI) complex in regulating the DNA-methylated SUPPRESSOR OF DRM1 DRM2 CMT3 (SDC) reporter. We demonstrate that components of the plant-specific ISWI complex, including CHROMATIN REMODELING PROTEIN11 (CHR11), CHR17, DDT-RELATED PROTEIN4 (DDR4), and DDR5, function to partially derepress silenced genes and transposable elements (TEs), through their function in regulating nucleosome distribution. This action also requires the known transcriptional activator DNAJ proteins, providing a mechanistic link between nucleosome remodeling and transcriptional activation. Genome-wide studies revealed that DDR4 causes changes in nucleosome distribution at numerous loci, a subset of which is associated with changes in DNA methylation and/or transcription. Our work reveals a mechanism for balancing transcriptional flexibility and faithful silencing of DNA-methylated loci. As both ISWI and MORC family genes are widely distributed across plant and animal species, our findings may represent a conserved eukaryotic mechanism for fine-tuning gene expression under epigenetic regulation.

Participation of nuclear genes in chloroplast gene expression

A genetic screen in Arabidopsis was developed to explore the regulation of chloroplast protein import in vivo using two independent reporters representing housekeeping and photosynthetic pre-proteins. We first used 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase*), a key enzyme in the shikimic acid pathway, with a mutation that confers tolerance to the herbicide glyphosate. Because the EPSP synthase* pre-protein must be imported for its function, the loss of glyphosate tolerance provided an initial indication of an import deficiency. Second, the fate of GFP fused to a ferredoxin transit peptide (FD5-GFP) was determined. A class of altered chloroplast import (aci) mutants showed both glyphosate sensitivity and FD5-GFP mislocalized to nuclei. aci2-1 was selected for further study. Yellow fluorescent protein (YFP) fused to the transit peptide of EPSP synthase* or the small subunit of Rubisco was not imported into chloroplasts, but also localized to nuclei during protoplast transient expression. Isolated aci2-1 chloroplasts showed a 50% reduction in pre-protein import efficiency in an in vitro assay. Mutants did not grow photoautotrophically on media without sucrose and were small and dark green in soil. aci2-1 and two alleles code for Moco-sulfurase, which activates the aldehyde oxidases required for the biosynthesis of the plant hormones abscisic acid (ABA) and indole-acetic acid (IAA) and controls purine nucleotide (ATP and GTP) turnover and nitrogen recycling via xanthine dehydrogenase. These enzyme activities were not detected in aci2-1. ABA, IAA and/or purine turnover may play previously unrecognized roles in the regulation of chloroplast protein import in response to developmental, metabolic and environmental cues.

Chapter 11 - RNA silencing as a general defence mechanism against pathogens

As simple as ACB – new insights into the role of acyl‐CoA‐binding proteins in Arabidopsis

Plant growth and development are dependent on chloroplast development and function. Constitutive high level accumulation of a chloroplast stress signal, 3′-phosphoadenosine-5′-phosphate (PAP), confers drought tolerance to plants, but slow downs and alters plant growth and development. PAP, a by-product of sulfur metabolism, is maintained at very low levels by the SAL1 phosphatase during vegetative growth of Arabidopsis and accumulates in rosettes during drought and excess light. Eight independent forward genetic screens in Arabidopsis identified SAL1 as the regulator of multiple phenotypes related to stress responses, hormonal signaling and/or perception. In this perspective article, we collate all the sal1 phenotypes published in the past two decades, and distill the different pathways affected. Our meta-analysis of publicly available sal1 microarray data coupled to preliminary hormonal treatment and profiling results on sal1 indicate that homeostasis and responses to multiple hormones in sal1 are altered during rosette growth, suggesting a potential connection between SAL1-PAP stress retrograde pathway and hormonal signaling. We propose the SAL1-PAP pathway as a case study for integrating chloroplast retrograde signaling, light signaling and hormonal signaling in plant growth and morphogenesis.