bs_bs_banner Environmental Microbiology (2013) 15(3), 716–735 doi:10.1111/1462-2920.12066 Genes expressed by the biological control bacterium Pseudomonas protegens Pf-5 on seed surfaces under the control of the global regulators GacA and RpoS Teresa A. Kidarsa,1 Brenda T. Shaffer,1 biosynthetic gene expression profiles. Future studies Neal C. Goebel,2 Daniel P. Roberts,3 evaluating biological control mechanisms can now Jeffrey S. Buyer,3 Aaron Johnson,4 focus on genes expressed by Pf-5 on seed surfaces, Donald Y. Kobayashi,5 T. Mark Zabriskie,2 the habitat where the bacterium interacts with seed- Ian Paulsen6 and Joyce E. Loper1* infecting pathogens to suppress seedling diseases. 1USDA-ARS Horticultural Crops Research Laboratory, Corvallis, OR, USA. Introduction 2Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR, USA. The soil bacterium Pseudomonas protegens Pf-5 (for- 3USDA-ARS Sustainable Agricultural Systems merly Pseudomonas fluorescens Pf-5; Ramette et al., Laboratory, Beltsville, MD, USA. 2011) colonizes the surfaces of seeds and roots and can 4J. Craig Venter Institute, Rockville, MD, USA. protect plants from infection by certain soil-borne fungal, 5Department of Plant Biology and Pathology, Rutgers oomycete and bacterial plant pathogens (Howell and University, New Brunswick, NJ, USA. Stipanovic, 1979; 1980; Xu and Gross, 1986; Kraus and 6Department of Chemistry and Biomolecular Sciences, Loper, 1992; Pfender et al., 1993; Rodriguez and Pfender, Macquarie University, Sydney, NSW, Australia. 1997; Sharifi-Tehrani et al., 1998). Secondary metabolite production is an important contributing factor for the bio- logical control of many plant diseases by Pseudomonas Summary spp. (Haas and Keel, 2003; Rezzonico et al., 2007; Gene expression profiles of the biological control Weller et al., 2007), and Pf-5 produces a spectrum of strain Pseudomonas protegens Pf-5 inhabiting pea secondary metabolites with antibiotic activity, including seed surfaces were revealed using a whole-genome 2,4-diacetylphloroglucinol (DAPG), pyoluteorin, pyrrolni- oligonucleotide microarray. We identified genes trin, hydrogen cyanide, rhizoxin analogues and orfamide expressed by Pf-5 under the control of two global A (Howell and Stipanovic, 1979; 1980; Nowak-Thompson regulators (GacA and RpoS) known to influence bio- et al., 1994; Gross et al., 2007; Loper et al., 2008). A logical control and secondary metabolism. Transcript similar suite of antibiotics is produced by the biological levels of 897 genes, including many with unknown control strain CHA0, which is closely related to Pf-5 (Haas functions as well as those for biofilm formation, cyclic and Keel, 2003). Antibiotic production by strains Pf-5 and diguanylate (c-di-GMP) signalling, iron homeostasis CHA0 is regulated at multiple levels, including by the and secondary metabolism, were influenced by one Gac/Rsm signal transduction pathway (Lapouge et al., s or both regulators, providing evidence for expression 2008) and the stationary-phase sigma factor RpoS (s ; of these genes by Pf-5 on seed surfaces. Comparison Sarniguet et al., 1995; Heeb et al., 2005). of the GacA and RpoS transcriptomes defined for Pf-5 In the Gac/Rsm signal transduction pathway, GacA acts grown on seed versus in broth culture overlapped, as a positive transcriptional regulator of genes encoding but most genes were regulated by GacA or RpoS the small regulatory RNAs, RsmX, RsmY and RsmZ under only one condition, likely due to differing levels (Heeb et al., 2002; Valverde et al., 2003; Kay et al., 2005). of expression in the two conditions. We quantified These small RNAs sequester the RNA-binding proteins secondary metabolites produced by Pf-5 and gacA RsmA and RsmE, relieving translational repression of and rpoS mutants on seed and in culture, and found genes having upstream binding sites for these repressor that production profiles corresponded generally with proteins (Valverde et al., 2003; Kay et al., 2005). GacA also has extensive effects on the transcriptome of Pseu- domonas spp.: transcript levels of nearly 10% of the Received 15 June, 2012; revised 20 October, 2012; accepted 22 November, 2012. *For correspondence. E-mail Joyce.Loper@ protein-encoding genes in the Pf-5 genome are influ- ars.usda.gov; Tel. (+1) 541 738 4057; Fax (+1) 541 738 4025. enced by GacA (Hassan et al., 2010), although this is Published 2012. This article is a U.S. Government work and is in the public domain in the USA. GacA and RpoS regulation of Pf-5 genes on seeds 717 likely due largely to indirect regulation via other genes and also may influence the expression of biocontrol traits under translational regulation by the Gac/Rsm pathway, by bacteria on the seed surface. Indeed, the availability as in Pseudomonas aeruginosa (Brencic and Lory, 2009). and composition of carbon and other nutrients are major Among the many genes regulated by GacA and its factors influencing antibiotic production by P. protegens cognate sensor kinase GacS are those required for pro- Pf-5 and CHA0 (Nowak-Thompson et al., 1994; Kraus duction of secondary metabolites and secreted exoen- and Loper, 1995; Duffy and Défago, 1999; Haas and Keel, zymes, such as chitinase and the AprA protease 2003; Haas and Défago, 2005; Valverde and Haas, 2008; (Lapouge et al., 2008; Hassan et al., 2010). Humair et al., 2009). The RpoS sigma factor directs RNA polymerase to pro- The primary focus of this work was to identify genes moters of certain genes transcribed in response to stress expressed under the control of GacA and RpoS by P. pro- or during the transition to stationary phase (Hengge- tegens Pf-5 inhabiting seed surfaces. Because both GacA Aronis, 2002), directly or indirectly regulating the expres- and RpoS influence biological control by Pseudomonas sion of a large set of genes having diverse functions spp., the GacA and RpoS transcriptomes of Pf-5 inhabit- (Schuster et al., 2004; Battesti et al., 2011). An rpoS ing the spermosphere are expected to include genes con- mutant of Pf-5 shows reduced tolerance to oxidative and tributing to biological control. We defined and compared osmotic stress, as well as to freezing, starvation, UV the GacA and RpoS transcriptomes of Pf-5 grown in irradiation and desiccation (Sarniguet et al., 1995; Stock- culture and on seed surfaces. While many genes were well and Loper, 2005). RpoS has differential effects on regulated by GacA and RpoS both in culture and on seed secondary metabolite production in Pf-5, with an rpoS surfaces, the majority of genes were regulated signifi- mutant of Pf-5 overproducing pyoluteorin and DAPG, cantly under only one condition, thus greatly expanding while failing to produce pyrrolnitrin (Pfender et al., 1993; the list of genes known to be under the control of these Sarniguet et al., 1995). global regulators. This study provides evidence for the Both the Gac/Rsm system and RpoS affect the ability of expression of hundreds of genes by Pf-5 inhabiting seed Pf-5 or CHA0 to act as biological control agents. gacS, surfaces and highlights GacA- and RpoS-regulated genes gacA, rsmX, rsmY or rsmZ mutants of CHA0 are deficient that could contribute to biological control of seed-infecting in biological control of several plant diseases (Schmidli- pathogens. Sacherer et al., 1997; Valverde et al., 2003; Zuber et al., 2003; Kay et al., 2005). An rpoS mutant of Pf-5 showed Results and discussion enhanced biological control of postemergence damping- off of cucumber caused by Pythium ultimum (Sarniguet The major goal of our study was to determine the effects et al., 1995), but a reduced ability to inhibit Pyrenophora of GacA and RpoS on gene expression of Pf-5 grown on tritici-repentis in wheat straw (Pfender et al., 1993). seed surfaces. We assessed the transcriptomes of Pf-5 Because the Gac/Rsm system and RpoS have such and gacA and rpoS mutants on surface-sterilized pea broad effects on the physiology of the bacterium, it is seed, as well as the chemical composition of the pea seed unclear whether their influence on biological control is exudate (Table S1). Initial experiments showed that the mediated solely through changes in secondary metabolite population size of Pf-5 increased rapidly in the first 24 h production versus other important traits, such as the following seed inoculation (from ~ 107 cfu per seed to ability to efficiently colonize seed or root surfaces. ~ 108 cfu per seed), whereas minimal additional growth To effect biological control of seed-infecting pathogens, occurred in the subsequent 24 h period. Our observation Pf-5 must express genes needed to suppress target that the population size of Pf-5 on seed surfaces reaches pathogens, as well as those required for growth and sur- carrying capacity at 24 h after the onset of seed imbibition vival in the spermosphere, the region of the soil under the is consistent with the temporal pattern of pea seed exu- influence of seeds. The main energy source in the sper- dation, as the highest levels of exudation occur during the mosphere is reduced carbon released from germinating initial hours after the onset of imbibition, with exudation seeds in the form of exudate (Nelson, 2004). Propagules diminishing 24 h after sowing (Roberts et al., 1999; 2000). of many fungal and oomycete plant pathogens can remain Preliminary reverse-transcriptase real-time PCR (RT- dormant in the soil for months or years but germinate qPCR) analysis showed that wild-type and mutant bacte- quickly in response to seed exudates, producing mycelia ria grown on seed surfaces for 24 h displayed expression that can infect seeds. Consequently, the spermosphere is differences in genes previously found to be regulated by often the initial point of interaction between pathogens GacA or RpoS in culture, including genes involved in the infecting seeds and biological control microorganisms and production of secondary metabolites (data not shown).