Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators

Abstract

To elucidate host processes and components required for the sustained growth and reproduction of the obligate biotrophic fungus Golovinomyces orontii on Arabidopsis thaliana , laser microdissection was used to isolate cells at the site of infection at 5 days postinfection for downstream global Arabidopsis expression profiling. Site-specific profiling increased sensitivity dramatically, allowing us to identify specific host processes, process components, and their putative regulators hidden in previous whole-leaf global expression analyses. For example, 67 transcription factors exhibited altered expression at the powdery mildew (PM) infection site, with subsets of these playing known or inferred roles in photosynthesis, cold/dehydration responses, defense, auxin signaling, and the cell cycle. Using integrated informatics analyses, we constructed putative regulatory networks for a subset of these processes and provided strong support for host cell cycle modulation at the PM infection site. Further experimentation revealed induced host endoreduplication occurred exclusively at the infection site and led us to identify MYB3R4 as a transcriptional regulator of this process. Induced endoreduplication was abrogated in myb3r4 mutants, and G. orontii growth and reproduction were reduced. This suggests that, by increasing gene copy number, localized endoreduplication serves as a mechanism to meet the enhanced metabolic demands imposed by the fungus, which acquires all its nutrients from the plant host.

Nuclear Function of Subclass I Actin-Depolymerizing Factor Contributes to Susceptibility in Arabidopsis to an Adapted Powdery Mildew Fungus

“…Previously identified Arabidopsis genes, the loss of whose expression causes enhanced resistance against adapted powdery mildew fungi, include POWDERY MILDEW RESISTANT1 (PMR1) to PMR6 (Vogel and Somerville, 2000;Vogel et al, 2002Vogel et al, , 2004Nishimura et al, 2003), MILDEW RESISTANCE LOCUS O2 (MLO2; Consonni et al, 2006), ENHANCED DISEASE RESISTANCE1 (EDR1), EDR2, and EDR3 (Frye and Innes, 1998;Frye et al, 2001;Tang et al, 2005aTang et al, , 2006, CONSTITUTIVE EXPRESSION OF VSP1 (CEV1; Ellis et al, 2002), PLANT UBIQUITIN REGULATORY X DOMAIN-CONTAINING PROTEIN2 (Chandran et al, 2009), FERONIA (FER; Kessler et al, 2010), MYB3R4 (Chandran et al, 2010), AUTOPHAGY-RELATED2 (ATG2), ATG5, ATG7, ATG10, and ATG18 (Wang et al, 2011a(Wang et al, , 2011b, PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE TYPE2C (PAPP2C; Wang et al, 2012), ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM1 (AMSH1; Katsiarimpa et al, 2013), Arabidopsis LIFEGUARD1 (AtLFG1) and AtLFG2 (Weis et al, 2013a), LESION INITIATION2 (LIN2; Guo et al, 2013), DP-E2F-LIKE1 (DEL1; Chandran et al, 2014), and CHYTOCHROME P450 83A1 (CYP83A1; Weis et al, 2013bWeis et al, , 2014. Mutations in PMR5, AtLFG6, CEV1, and CYP83A1 cause modifications of cuticular wax or cell wall structure, negatively affecting the infection of powdery mildew fungi that grow and proliferate on the leaf surface.…”

Jasmonate Controls Leaf Growth by Repressing Cell Proliferation and the Onset of Endoreduplication while Maintaining a Potential Stand-By Mode

“…Recently, the occurrence of endoreduplication has been associated with an increased potential for further cell functions , such as the capacity for future cellular growth (Breuer et al, 2010), the regulation of responses against plant pathogen attacks (Chandran et al, 2010;Wildermuth, 2010), or a role in the maintenance of cell fate (Bramsiepe et al, 2010). In terms of cellular machinery, although basic plant cell cycle and growth regulators have become increasingly well characterized during Arabidopsis development (Gutierrez, 2009;Boruc et al, 2010;Van Leene et al, 2010, 2011, little is known about their precise regulation/ coordination.…”

Why do plants need so many cyclin-dependent kinase inhibitors?

“…Early hints of a connection between the cell cycle and pathogen responses came from the observation that either genetic manipulation of plant defense pathways 30 or pathogen infection 31 can trigger endoreplication. More recent work shows that smr1 mutants have increased pathogen susceptibility 21 and that both SMR1 and KRP2 play roles in Arabidopsis effector-triggered immunity to bacterial and fungal pathogens through a physical interaction with a nuclear envelope protein CONSTITUTIVE EXPRESSOR OF PATHO-GENESIS-RELATED GENES5 (CPR5), apparently by contributing to the hyperphosphorylation of the key cell cycle regulator RETI-NOBLASTOMA-RELATED1 (RBR1).…”