Targeted promoter editing for rice resistance to Xanthomonas oryzae pv. oryzae reveals differential activities for SWEET14‐inducing TAL effectors

Abstract

SummaryAs a key virulence strategy to cause bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo) injects into the plant cell DNA‐binding proteins called transcription activator‐like effectors (TALEs) that bind to effector‐binding elements (EBEs) in a sequence‐specific manner, resulting in host gene induction. TALEs AvrXa7, PthXo3, TalC and Tal5, found in geographically distant Xoo strains, all target OsSWEET14, thus considered as a pivotal TALE target acting as major susceptibility factor during rice–Xoo interactions. Here, we report the generation of an allele library of the OsSWEET14 promoter through stable expression of TALE‐nuclease (TALEN) constructs in rice. The susceptibility level of lines carrying mutations in AvrXa7, Tal5 or TalC EBEs was assessed. Plants edited in AvrXa7 or Tal5 EBEs were resistant to bacterial strains relying on the corresponding TALE. Surprisingly, although indels within TalC EBE prevented OsSWEET14 induction in response to BAI3 wild‐type bacteria relying on TalC, loss of TalC responsiveness failed to confer resistance to this strain. The TalC EBE mutant line was, however, resistant to a strain expressing an artificial SWEET14‐inducing TALE whose EBE was also edited in this line. This work offers the first set of alleles edited in TalC EBE and uncovers a distinct, broader range of activities for TalC compared to AvrXa7 or Tal5. We propose the existence of additional targets for TalC beyond SWEET14, suggesting that TALE‐mediated plant susceptibility may result from induction of several, genetically redundant, host susceptibility genes by a single effector.

Sugar flux and signaling in plant–microbe interactions

“…Mutations involving the EBE reduce or eliminate effector binding, preventing SWEET gene induction. TALEN‐mediated deletions directed at OsSWEET14 and, more recently, CRISPR‐Cas9‐mediated mutations at OsSWEET13 have produced plants that are resistant to strains of Xoo (Li et al ., ; Zhou et al ., ; Blanvillain‐Baufumé et al ., ). Alteration of promoter mutations, either present in natural variants of OsSWEET11/ Xa13 as well as those obtained by genome editing, prevent binding of the TAL effectors and their usurpation of SWEET gene regulation in infected cells, which leads to a recessive ‘gain of function’ resistance that does not noticeably impair normal SWEET function, and importantly has no negative effects on yield potential (Chen et al ., ).…”

An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly

“…Such important TALes represent attractive targets to create disease resistance in host crop plants by interfering with the disease mechanism (Schornack et al ., ). Naturally occurring or gene editing‐derived alleles with EBE variations, if disruptive to TALe binding, in the promoters of S genes targeted by TALes confer genetically recessive resistance (Blanvillain‐Baufumé et al ., ; Chu et al ., ; Li et al ., ; Liu et al ., ). Multiplex genome editing produced single engineered rice lines that carried multiple mutations in three SWEET gene promoters and resulted in broad‐spectrum blight resistance (R. Oliva et al ., unpublished data).…”

A common metabolomic signature is observed upon inoculation of rice roots with various rhizobacteria

“…Real time PCR conditions were: a denaturation stage of 10 min at 95°C, an amplification stage of 45 cycles of 15 sec at 95°C, 10 sec at the specific annealing temperature (Table S2) and 20 sec at 72°C, and a melting curve stage of 5 sec at 97°C and 1 min at 65°C increased to 97°C with a ramp rate of 0.5°C · s –1 . To normalize data, two reference genes whose expression has been confirmed to be stable in our experimental conditions (data not shown) were used: Os03g0177400 encoding elongation factor 1A (Blanvillain‐Baufumé et al ) and Os06g0215200 encoding a Zinc finger, U1‐C type domain containing protein (Narsai et al ). The level of gene expression was compared to the non‐inoculated condition using the Pfaffl method 2 ‐ΔΔCt (Livak and Schmittgen, ) corrected by each primer pair efficiency.…”