ORIGINAL ARTICLE Maize host requirements for Ustilago maydis tumor induction Virginia Walbot �� David S. Skibbe Received: 5 July 2009 / Accepted: 27 July 2009 / Published online: 19 August 2009 �� Springer-Verlag 2009 Abstract The biotrophic pathogen Ustilago maydis cau- ses tumors by redirecting vegetative and floral develop- ment in maize (Zea mays L.). After fungal injection into immature tassels, tumors were found in all floral organs, with a progression of organ susceptibility that mirrors the sequential location of foci of cell division in developing spikelets. There is sharp demarcation between tumor- forming zones and areas with normal spikelet maturation and pollen shed within and immediately adjacent to the tumor zone, developing anthers often emerge precociously and exhibit a range of developmental defects suggesting that U. maydis signals and host responses are restricted spatially. Male-sterile maize mutants with defects in anther cell division patterns and cell fate acquisition prior to meiosis formed normal adult leaf tumors, but failed to form anther tumors. Methyl jasmonate and brassinosteroid phe- nocopied these early-acting anther developmental mutants by generating sterile zones within tassels that never formed tumors. Although auxin, cytokinin, abscisic acid and gib- berellin did not impede tassel development, the Dwarf8 mutant defective in gibberellin signaling lacked tassel tumors the anther ear1 mutant reduced in gibberellin content formed normal tumors and Knotted1, in which there is excessive growth of leaf tissue, formed much larger vegetative and tassel tumors. We propose the hypothesis that host growth potential and tissue identity modulate the ability of U. maydis to redirect differentiation and induce tumors. Keywords Anther Brassinosteroid Male-sterile Methyl jasmonate Ustilago maydis Introduction Although the biotrophic fungal pathogen Ustilago maydis grows on many flowering plants (Leon-Ram��rez �� �� et al. 2004), it exhibits exquisite host specificity in eliciting tumors. This capacity is restricted to domesticated maize (Zea mays L.) and its wild progenitor teosinte (Banuett 2002). In natural infections, haploid fungal sporidia elon- gate on maize surfaces, complementary mating types fuse to form a dikaryon, and the resulting filament enters the plant by direct penetration. Fungal hyphae proliferate rapidly in intimate contact with invaginations of the host plasma membrane within the infection zone (Doehlemann et al. 2009). Unlike oncogenic agents in animals that reactivate cell division, tumors induced by U. maydis require dividing host cells, subverting controls on normal maize cell proliferation to promote excessive cell division, polyploidization and enormous cell expansion (Callow and Ling 1973 Callow 1975 Banuett 2002). Within tumors, diploid fungal cells mature into teliospores, the primary agent of fungal dispersal. Altogether, fungal infection, growth and spore differentiation require about 2 weeks and culminate in the release of billions of diploid teliospores (Banuett and Herskowitz 1996). Based on gene deletion assays and analysis of the U. maydis genome, the current hypothesis to explain tumor induction is that this pathogen secretes a suite of effector proteins that trigger abnormal host cell differentiation. Communicated by Sheila McCormick. V. Walbot (&) D. S. Skibbe Department of Biology, Stanford University, Stanford, CA 94305-5020, USA e-mail: walbot@stanford.edu D. S. Skibbe e-mail: skibbe@stanford.edu 123 Sex Plant Reprod (2010) 23:1���13 DOI 10.1007/s00497-009-0109-0

Tumor expansion undoubtedly depends on plant hormones. Although U. maydis produces significant amounts in planta, fungal-derived auxin is not essential for pathoge- nicity (Kamper �� et al. 2006 Reineke et al. 2008). Recent sequencing of the U. maydis genome demonstrated that this pathogen encodes 386 predicted secreted proteins, some of which are required for inciting seedling leaf tumors by unknown mechanisms (Mueller et al. 2008). Maize and other flowering plants encode hundreds of predicted membrane-localized receptor kinases, but only a few have defined ligands. These and other poorly defined systemic and cell-to-cell signaling networks in maize are likely to be targets of U. maydis proteins that redirect host development leading to tumorogenesis. To date, the requirements for tumor formation have been investigated by manipulating the pathogen, using the host plant, typically a seedling, as a substrate. Therefore, many basic questions on tumor growth have not been explored from the plant perspective. For example, are specific plant genes required for tumor formation in different plant organs? Would maize mutants altered in hormone biosyn- thesis, perception, or signal transduction form larger or fewer tumors? To explore these and related questions, we have surveyed the impact of plant hormones and maize mutants defective in growth control. The tassel was chosen because it can be carefully staged, and it has a well- established progression of foci of cell division. The tassel is a complex inflorescence containing well-synchronized cohorts of developing flowers that collectively span about 1 week of floral development. Additionally, numerous male-sterile (ms) mutants have defects that disrupt the cell division patterns and acquisition of cell fate in anthers, providing the opportunity to explore whether U. maydis can elicit tumors in host organs with altered development. Collectively, these studies initiate a genetic analysis of the host requirements for tumor formation. Materials and methods Plant material and growth conditions Inbred line stocks are maintained by the Walbot Laboratory and include A619, B73, Ky21 and W23 (recessive for bz2, a gene in the anthocyanin biosynthetic pathway). Other Walbot Laboratory stocks include the homozygous an1 bz2 deletion mutant in W23, and the ms lines msca1, mac1 and ms26 backcrossed twice into W23 and maintained as 1:1 segregating families. Kn1-OL (segregating 1:1 nor- mal:mutant), was obtained from Sarah Hake (Plant Gene Expression Center, USDA, Albany CA). The spi1 stock (segregating 1:1 normal:mutant) was obtained from Paula McSteen (Pennsylvania State University, University Park PA). The Dwarf8 stock was obtained from the Maize Genetics Cooperation Stock Center (http://maizecoop. cropsci.uiuc.edu/ and maintained by crossing onto inbred lines to generate families segregating 1:1 normal:mutant. The ms and inbred lines were grown outdoors in sum- mer 2007 at Stanford CA. Families of 25 (uniform) or 50 (segregating stocks) were planted weekly for U. maydis injections to define the period of tumor susceptibility in specific tassel structures primarily, data from W23 are reported, but the experiments also included Ky21, A619 and B73, all of which gave similar results. In summer 2008, these inbred lines were grown again for evaluation of hormone treatments. The W23 bz2 tester line was grown in multiple plantings in a greenhouse equipped with lights providing *50% of summer noon solar fluence (16-h light/ 8-h dark) at Stanford CA. A subset of inbred and ms lines were grown under these conditions, as well as all of the developmental mutants. Greenhouse families typically contained 36���40 individuals in uniform stocks and 80 individuals in segregating stocks grown in two ranks of pots with automatic watering and fertilizer treatments with cohorts of 8���10 individuals used in each treatment. There was a slight, but consistent, temperature gradient across the greenhouse space resulting in a 1���2 days of developmental tassel stage difference between the first (advanced) and last (slowest) plants. To minimize age effects, in experiments with two or more treatments, every nth plant down a row was assigned to the same treatment cohort. Ustilago maydis culture and injections The FB1 and FB2 haploid mating types of U. maydis were obtained from Flora Banuett (Long Beach State University, Long Beach, CA) and grown as described in Banuett and Herskowitz (1994, 1996). Briefly, strains were streaked out separately on solid media and grown for 2 days at 27���28��C. Individual colonies were picked and streaked out for a second 2-day growth period. From these second plates, a loop full of FB1 or FB2 was inoculated into 15��� 25-mL liquid media in a 125-mL Erlenmeyer flask for growth at 27���28��C on a rotary shaker set at 125 rpm for 16���18 h. To initiate fungal fusion, the overnight liquid FB1 and FB2 cultures were mixed, and within 45 min injected into plants. In the experiments involving maize hormone mutants, the solopathogenic strain SG200 (supplied by Regine Kahmann, Max Planck Institut, Marburg Germany) was used (Kamper �� et al. 2006) the strain was grown fol- lowing the procedures of Kruger �� et al. (2000). Cells were collected by centrifugation at 3,0009g for 15 min, and then resuspended in sterile water at A600nm = 1.0 prior to injection. Injections were performed using 3 or 12-mL sterile plastic syringes with 25 or 26 gauge needles. Typically, 2 Sex Plant Reprod (2010) 23:1���13 123