How the plasma membrane senses external heat-stress signals to communicate with chloroplasts to orchestrate thermotolerance remains elusive. We identified a quantitative trait locus, Thermo-tolerance 3 (TT3), consisting of two genes, TT3.1 and TT3.2, that interact together to enhance rice thermotolerance and reduce grain-yield losses caused by heat stress. Upon heat stress, plasma membrane-localized E3 ligase TT3.1 translocates to the endosomes, on which TT3.1 ubiquitinates chloroplast precursor protein TT3.2 for vacuolar degradation, implying that TT3.1 might serve as a potential thermosensor. Lesser accumulated, mature TT3.2 proteins in chloroplasts are essential for protecting thylakoids from heat stress. Our findings not only reveal a TT3.1-TT3.2 genetic module at one locus that transduces heat signals from plasma membrane to chloroplasts but also provide the strategy for breeding highly thermotolerant crops.